Quantitative multiplex methylation-specific PCR

ABSTRACT

Methods are provided for diagnosing in a subject a condition, such as a carcinoma, sarcoma or leukemia, associated with hypermethylation of genes by isolating the genes from tissue containing as few as 50 to 1000 tumor cells. Using quantitative multiplex methylation specific PCR (QM-MSP), multiple genes can be quantitatively evaluated from samples usually yielding sufficient DNA for analyses of only 1 or 2 genes. DNA sequences isolated from the sample are simultaneously co-amplified in an initial multiplex round of PCR, and the methylation status of individual hypermethylation-prone gene promoter sequences is then determined separately or in multiplex using a real time PCR round that is methylation status-specific. Within genes of the panel, the level of promoter hypermethylation as well as the incidence of promoter hypermethylation can be determined and the level of genes in the panel can be scored cumulatively. The QM-MSP method is adaptable for high throughput automated technology.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application claims the benefit of priority under 35 U.S.C. §119(e)of U.S. Ser. No. 60/515,100, filed Oct. 28, 2003, the entire contents ofwhich are incorporated herein by reference.

GRANT INFORMATION

This invention was made with Government support under DAMD 17-01-1-0286,awarded by the Department of Defense, and the NIH P50CA88843 awarded bythe National Cancer Institute. The government may have certain rights inthe invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to cancer markers and more specificallyto a method of evaluating gene methylation within a sample, referred toherein as quantitative multiplex methylation-specific PCR (QM-MSP), inorder to detect conditions associated with methylation status of genes.

2. Background Information

Epigenetic alterations including hypermethylation of gene promoters areproving to be consistent and early events in neoplastic progression(Hanahan D. and Weinberg R. A. Cell (2000) 100:57-70; Wamecke P. M. andBestor T. H. Cum Opin Oncol (2000) 12:68-73; Yang X. et al. Endocr RelatCancer (2001) 8:115-127; and Widschwendter M. and Jones P. A. Oncogene(2002) 211:5462-5482). Such alterations are thought to contribute to theneoplastic process by transcriptional silencing of tumor suppressor geneexpression, and by increasing the rate of genetic mutation (Wajed S. A.et al. Ann Surg (2001) 234:10-20 and Jones P. A. and Baylin S. B. NatRev Genet (2002) 3: 415-428). DNA methylation is reversible, since itdoes not alter the DNA sequence; however, it is heritable from cell tocell. Methylated genes can serve as biomarkers for early detection ofcancer for risk assessment and for predicting response to therapy.

Widschwendter and Jones (supra) reviewed over 40 genes whose expressionis lost in breast cancer due to promoter hypermethylation, and othershave studied hypermethylation of genes including NES-1 (Goyal J. et al.Cancer Res (1998) 58:4782-4786; Dhar S. et al. Clin Cancer Res (2001)7:3393-3398; Li B. et al. Cancer Res (2001) 61:8014-8021; and Yunes M.J. et al. Int J Radiat Oncol Biol Phys (2003) 56:653-657) APC (KashiwabaM. et al. J Cancer Res Clin Oncol (1994) 120:727-731; Virmani A. K. etal. Clin Cancer Res (2001) 7:1998-2004; and Sarrio D. et al. Int JCancer (2003) 106:208-215), Cyclin D2 (Evron E. et al. Cancer Res (2001)61:2782-2787 and Lehmann U. et al. Am J Pathol (2002) 160:605-612), RARB(Widschwendter M. et al. J Natl Cancer Inst (2000) 92:826-832; Yan L. etal. J Mammary Gland Biol Neoplasia (2001) 6:183-192; and Sirchia S. M.et al. Cancer Res (2002) 62: 2455-2461), TWIST (Evron E. et al. Lancet(2001) 357:1335-1336), RASSF1A (Lehmann U. et al. Cancer Res (2001)61:8014-8021; Burbee D. G. et al. J Natl Cancer Inst (2001) 93:691-699;and Dammann R. et al. Cancer Res (2001) 61:3105-3109), and HIN1 (Krop I.E. et al. Proc Natl Acad Sci USA (2001) 98:9796-9801) in tissue, bloodand ductal fluids. Since methylation changes often appear early indisease, detection of hypermethylated genes could identify tissuesderived from subjects with increased risk. Furthermore, the reversiblenature of methylation offers the potential to revert aspects of thecancer phenotype with the appropriate therapy (Fackler M. J. et al. JMammary Gland Biol Neoplasia (2003) 8:75-89).

Tumor DNA can be found in various body fluids and these fluids canpotentially serve as diagnostic material. Evaluation of tumor DNA inthese fluids requires methods that are specific as well as sensitive.For instance, a PCR-base technique called methylation-specific PCR (MSP)is reported to detect one copy of methylated genomic DNA in one-thousandunmethylated copies of genomic DNA (Herman J. G. et al. Proc Natl AcadSci U S A (1996) 93:9821-9826). This approach has been modified in orderto co-amplify several genes simultaneously in a nested or multiplex MSPassay (Palmisano W. A. et al. Cancer Res (2000) 60:5954-5958; Buller A.et al. Mol Diagn (2000) 5:239-243; and Brock M. V. et al. Clin CancerRes (2003) 9:2912-2919). The read out is gel-based and qualitative (“allor nothing”). This method has been used to establish the frequency ofgene promoter hypermethylation among subjects with bronchial andesophageal carcinoma. However, the method cannot quantitatively measurethe levels of gene methylation.

Quantitative real time PCR (Q-PCR) allows a highly sensitivequantification of transcriptional levels or levels of the DNA of thegene of interest in a few hours with minimal handling the samples (HeidC. A. et al. Genome Res (1996) 6:986-994 and Gibson U. E. et al. GenomeRes (1996) 6:995-1001). cDNA or genomic copies of the gene of interestare quantitated by detecting PCR products as they accumulate using anoptically detectable polynucleotide probe. This technique is widelyused.

Quantitative MSP (Q-MSP) allows highly sensitive detection of genepromoter methylation levels by real time PCR with methylation-specificprimers probes (Lo Y. M. et al. Cancer Res (1999) 59:3899-3903; Trinh B.N. Methods (2001) 25:456-462; Wong I. H. et al. Clin Cancer Res (2003)9:1047-1052).

The advantage of fluorogenic probes over DNA binding dyes (e.g. sybergreen for real time PCR) is that specific hybridization between probeand target is required to generate fluorescent signal. Thus, withfluorogenic probes, non-specific amplification due to mis-priming orprimer-dimer artifact does not generate signal. However, Q-MSP analysisof multiple genes requires additional quantities of template DNA.Therefore, new and better methods are needed to increase the amount ofavailable DNA and to quantitate detection of gene methylation status forseveral genes.

SUMMARY OF THE INVENTION

The invention is based on the discovery of a method for evaluating thedegree of gene methylation within a single DNA sample, referred toherein as quantitative multiplex methylation-specific PCR (QM-MSP), toco-amplify many genes from amounts of sample previously used for justone or two genes. The invention provides a basis to define the extent ofgene promoter hypermethylation across a panel of genes, enhancing thediscriminatory power of this test to distinguish between normal andaltered condition(s). Determination of hypermethylation as compared withnormal methylation of samples may help to stratify different types orstages of conditions, such as cancer, associated with genehypermethylation. The QM-MSP method is based on real-time PCR that usesone or more distinguishable optically detectable probes to increase theassay specificity and the sensitivity such that one to ten copies of thedesired methylated gene among 100,000 unmethylated copies of the genecan be detected (Fackler M. J. et al. Cancer Res (2004) 64:4442-4452).QM-MSP is more economical in time and materials, is more informative,quantitative, and suitable for clinical format than MSP, multiplex MSPor Q-MSP. The invention methods solve the dilemma of how best todistribute the available DNA to allow robust quantitative analyses ofmany different genes from precious small samples.

In one embodiment, the invention provides methods for determining themethylation status of DNA in a sample by co-amplifying a plurality ofDNA sequences using a mixture of DNA sequence-specific, methylationstatus-independent outer primer pairs that selectively hybridize totheir cognate DNA sequences under conditions that allow generation of afirst amplification product containing first amplicons. Real time PCR isthen used to amplify unmethylated and/or methylated DNA from the firstamplicons under conditions that allow generation of second amplificationproducts for one or more genes, using two sets of primers comprising aDNA sequence-specific, methylation status-dependent (unmethylated ormethylated) inner primer pair and a (unmethylated or methylated) DNAsequence-specific probe. Second amplicons are detected by using one ormore distinguishable optically detectable labels per reaction. Acombination of inner primer pair and probe selectively hybridizes to onefirst amplicon, and the sets of inner primer pairs and probescollectively hybridize to a plurality of first amplicons in the firstamplification product. Signal intensities of the one or moredistinguishable labels in the second amplification products are detectedto determine the amount of methylation in a gene. A combined methylationvalue is derived for the methylation in the DNA sequences in the testsample from amounts of methylation determined for the secondamplification products across a panel of genes. For the purposes ofdetermining whether the gene amplicon is hyper- or hypo-methylated, thecombined methylation value of the test sample is compared with thecombined methylation value in a series of comparable normal DNA samples.

In yet another embodiment, the invention provides methods of diagnosingdevelopment of a condition associated with aberrant methylation of DNAin tissue of a subject by co-amplifying a plurality of DNA sequencesusing a mixture of DNA sequence-specific, methylation status-independentouter primer pairs that selectively hybridize to their cognate DNAsequences under conditions that allow generation of a firstamplification product containing first amplicons. Real time PCR is thenused to amplify unmethylated and/or methylated DNA from the firstamplicons under conditions that allow generation of second amplificationproducts for one or more genes, using for a gene two sets primerscomprising a DNA sequence-specific, methylation status-dependent(unmethylated or methylated) inner primer pair and a (unmethylated ormethylated) DNA sequence-specific probe. Second amplicons are detectedby using one or more distinguishable optically detectable labels perreaction. A combination of inner primer pair and probe selectivelyhybridizes to one first amplicon, and the sets of inner primer pairs andprobes collectively hybridize to a plurality of first amplicons in thefirst amplification product. Signal intensities of the one or moredistinguishable labels in the second amplification products are detectedto determine the amount of methylation in a gene. A combined methylationvalue is derived for the methylation in the DNA sequences in the testsample from amounts of methylation determined for the secondamplification products across a panel of genes. For the purposes ofdetermining whether the gene amplicon is hyper- or hypo-methylated, thecombined methylation value of the tissue of the subject is compared withthe combined methylation value in a series of comparable normal DNAsamples to diagnose the state of development of the condition in thesubject.

In still another embodiment, the invention provides methods fordiagnosing development of a cancer associated with hypermethylation ofCpG island DNA in carcinoma-associated tissue of a subject byco-amplifying CpG islands in a subject sample comprising severaldifferent DNA sequences isolated from the carcinoma-associated tissueusing a mixture of DNA sequence-specific, methylation status-independentouter primer pairs that selectively hybridize to one or more of the DNAsequences under conditions that allow generation of a firstamplification product containing first amplicons. Then, real time PCR isused to co-amplify the CpG islands in the first amplicons underconditions that allow generation of one or more second amplificationproducts, using one or more members of a set of DNA sequence-specificprobes comprising distinguishable fluorescent moieties and one or moremembers of a set of DNA sequence-specific methylation status-dependentinner primer pairs that selectively hybridize to one or more firstamplicon, wherein the sets of probes and inner primer pairs collectivelyhybridize to a plurality of different first amplicons in the firstamplification product. Fluorescence due to the presence ofdistinguishable fluorescent moieties in the second amplificationproducts are detected to determine the amount of methylation of the CpGislands therein; and a combined methylation value for the methylation inthe tissue of the subject is derived from the amounts of methylationdetermined for the second amplification products as compared with acombined methylation value in comparable normal tissue to diagnose thestate of development of the cancer in the subject.

In yet another embodiment, the invention provides methods for diagnosingthe state of development of a condition associated with hypermethylationof a gene in a mammalian subject by co-amplifying a plurality ofdifferent DNA sequences isolated from tissue of the subject associatedwith the condition using a mixture of DNA sequence-specific, methylationstatus-independent outer primer pairs that selectively hybridize to oneor more of the DNA sequences under conditions that allow generation of afirst amplification product containing first amplicons. Real time PCR isthen used to co-amplify the first amplicons under conditions that allowgeneration of one or more second amplification products using one ormore members of a set of DNA sequence-specific probes comprisingdistinguishable optically detectable labels and one or more members of aset of DNA sequence-specific methylation status-dependent inner primerpairs that selectively hybridize to a cognate first amplicon, whereinthe sets of probes and inner primer pairs collectively amplify aplurality of different first amplicons in the first amplificationproduct. Signal intensity of the distinguishable labels in the secondamplification products is detected to determine the amount ofmethylation of the second amplification products. A combined methylationvalue is derived for the methylation in the DNA sequences from amountsof methylation determined for the second amplification products in theDNA of the subject as compared with the combined methylation value in acomparable normal DNA sample to indicate the state of development of thecondition in the subject.

In another embodiment, the invention provides kits for determining themethylation status of a plurality of DNA sequences in a DNA sample. Theinvention kits include a set of DNA sequence-specific, methylationstatus-independent outer primer pairs that selectively hybridize tocognate DNA sequences under conditions that allow generation of a firstamplification product containing first amplicons; a set of DNAsequence-specific, methylation status-dependent inner primer pairs; anda set of DNA sequence-specific probes with one or more distinguishableoptically detectable labels. A combination of inner primer pair andprobe selectively hybridizes to one or more first amplicon, and the setsof inner primer pairs and probes collectively hybridize to a pluralityof first amplicons in the first amplification product.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic representation of the protocol for quantitativemultiplex methylation-specific PCR (QM-MSP) as used in the inventionmethods. In reaction #1 (RXN 1) a cocktail of gene-specific primer pairsis used to co-amplify DNA for a plurality of genes independent of theirDNA methylation status. In reaction #2 (RXN 2) quantitative real-timePCR is performed with gene-specific primers using the DNA templatederived from RXN 1 (diluted between 1:5 to 1:10⁴). Unmethylated (U) andmethylated (M) DNA were quantitated separately using methylation-statusspecific primers and a probe conjugated with the 6FAM label and TAMRAquencher. The probe is progressively degraded with each cycle of PCR andthe fluorescence signal generated by FAM is directly proportional to theextent of DNA amplification. To perform methylation analyses on twogenes simultaneously, RXN 2 also can contain a second set ofgene-specific internal methylation-dependent primers in combination witha probe conjugated with a different label, such as VIC™, as indicated bythe schematic and amplification plots for U RASSF1A and U RARβ (FIG.1B).

FIGS. 2A-B are graphs showing validation of QM-MSP using RASSF1Aunmethylated (U) and methylated (M) primer sets to amplify seriallydiluted fragments of multiplexed standard stock HSD/231 DNA. The cyclethreshold (C_(T)) was determined for each dilution of DNA. FIG. 2A showsa plot of ΔC_(T) (ΔC_(T)=C_(T)M−C_(T)U) versus dilution. FIG. 2B showsstandard curve plots (C_(T) VS Quantity) of serially diluted DNA. Aslope of −3.33 reflects a 2-fold amplification of DNA per cycle. Thecorrelation coefficient (R2) shows linearity (0.999) over the range ofDNA concentrations.

FIGS. 3A-B are graphs showing PCR cycles (X axis) plotted against thefluorescence intensity of the probe (Y axis) for multiplexed human spermDNA (HSD) (FIG. 3B) or MDA-MB231 (FIG. 3A) template tested withunmethylated (U) and methylated (M) primers for RASSF1A. Theunmethylated and methylated reactions were 100% specific since U primersdid not cross-react with MDA-MB231 DNA (100% methylated) and M primersdid not cross-react with HSD (100% unmethylated) DNA.

FIGS. 4A-B are graphs showing PCR cycles (X axis) plotted against thefluorescence intensity of the probe (Y axis) indicating detection ofhypermethylated genomic RASSF1A DNA in 1500-fold excess of unmethylatedDNA. Mixed, bisulfite-treated genomic HSD and 231 DNA (FIG. 4A: 600 pgHSD DNA and 40 pg MDA-MB231 DNA; FIG. 4B: 60 ng HSD DNA and 40 pg 231DNA) were subjected to QM-MSP.

FIGS. 5A-B are scatter plots show the level of gene promoterhypermethylation in normal and malignant breast tissues as determined bythe invention methods employing QM-MSP on normal tissues RASSF1A (n=28),TWIST (n=18) (FIG. 5A), Cyclin D2 (n=16), and HIN1 (n-14) (FIG. 5B) andDNA derived from individuals with invasive ductal carcinoma RASSF1A(n=19), TWIST (n=21), Cyclin D2 (n=21), and HIN1 (n=21) compared toleukocyte (Leuk) DNA derived from normal individuals (n=25). Displayedis the Ln_(e) (% M+1). A bar indicates the median of each group.

FIGS. 6A-B are bar graphs showing cumulative promoter hypermethylationof RASSF1A, TWIST, Cyclin D2, and HIN1 in normal and malignant breasttissues as determined by the invention methods as described in Table 5.FIG. 6A represents cumulative methylation in normal mammoplasty andtumor in a subgroup of samples from FIG. 5 where results were availablefor all four genes in the panel, normal (Mam; n=9) and tumor (n=19) werescored for cumulative methylation by adding the % M for all four geneswithin each sample. A maximum of 400 relative methylation units waspossible (e.g. MDA-MB231 control DNA is 100% methylated for each of thefour genes). The bar height reflects total cumulative methylation, whilethe segments correspond to the relative amounts of methylation of eachgene indicated. FIG. 6B shows mean cumulative methylation in normalmammoplasty versus tumor as determined by the unpaired t test(untransformed p=0.0002; transformed p=0.0001). Plotted is the mean(±standard error of the mean) amount of cumulative methylation.

FIGS. 7A-B are bar graphs showing cumulative promoter hypermethylationof RASSF1A, TWIST, Cyclin D2, and HIN1 in adjacent normal and malignantbreast tissues as described in Table 6. FIG. 7A shows the cumulativemethylation in paired samples of adjacent normal and tumor tissue (n=6)as quantitated by QM-MSP. Total possible units=400 (4 genes×100%). Themean of normal mammoplasty (n=9) samples (Mam) from FIG. 6B is shown atleft. FIG. 7B shows mean cumulative methylation in adjacent normalversus tumor (compared to normal mammoplasty). Differences betweennormal mammoplasty samples (median=0) and adjacent normal tissue samples(median=9 units) were significant (p=0.01; based on the Mann-Whitneytest). Plotted is the mean (±standard error of the mean) amount ofcumulative methylation found above in adjacent normal tissues (mean11.7±4.07) and the nearby tumor (mean 129±39.9), compared to normalmammoplasty (FIG. 7B) (mean 2.61±2.05; as shown in FIG. 6B). Thepercentage of methylation by gene as well as the extent of cumulativemethylation is indicated in the table (FIG. 7C) below the bar graphs.

FIG. 8 is a bar graph showing cumulative promoter hypermethylation inductal cells obtained by ductal lavage for RARB, RASSF1A, TWIST, CyclinD2, and HIN1 genes determined using the invention methods as describedin Table 7. Shown is the cumulative methylation of 50-1000 cells foundin cytologically benign (n=7) and pre-invasive (DCIS; n=4) samples. Foreach patient sample the cytology was graded as having mild, moderate ormarked atypia, or was found inadequate if fewer than 10 epithelial cellswere observed. The relative level of methylation of each gene isindicated by the height of the bar. Total possible units=500 (5genes×100%).

FIG. 9 is a bar graph showing cumulative promoter hypermethylation inductal cells obtained within nipple aspiration fluid for RARβ, RASSF1A,TWIST, Cyclin D2, HIN1, and ESR1 genes. The cumulative methylation ofthe 50-1000 cells is indicated. Cells showed either mild atypia (n=3) orappeared malignant (n=1) by cytology. The relative level of methylationof each gene is indicated by the height of the bar. Total possibleunits=600 (6 genes×100%).

FIG. 10 is a bar graph showing cumulative promoter hypermethylation inserum derived from patients (n=5) with metastatic breast cancer. Shownis the cumulative methylation of RARβ, RASSF1A, TWIST, Cyclin D2, HIN1,and ESR1 genes. The relative level of methylation of each gene isindicated by the height of the bar. Total possible units=600 (6genes×100%).

FIGS. 11A-B show the level of gene promoter hypermethylation in DNA froma series of patients (n=10) at high risk of developing breast cancer,but who are not know to have cancer. Paired samples of DNA isolated fromserum and from cells obtained by fine needle aspiration (FNA) and ductallavage (DL) (50-1000 cells) of each patient were analyzed by QM-MSP.FIG. 11A shows the cumulative methylation of RARβ, RASSF1A, TWIST,Cyclin D2, HIN1, ESR1, APC1, BRCA1 and BRCA2 genes. The relative levelof methylation of each gene is indicated by the height of the bar. Totalpossible units=900 (9 genes×100%). FIG. 11B shows the percentage ofmethylation of HIC1 determined by QM-MSP in the patient samples. Genepromoter hypermethylation of HIC1 was found to be significantly higherin FNA and DL samples than for other genes within the panel. For thisreason, HIC1 was analyzed separately from the other genes rather thaninclude HIC1 in the cumulative total for each patient.

FIG. 12 is a bar graph showing the level of gene promoterhypermethylation in cells derived from ductal lavage fluid from a seriesof patients (n=7) at high risk of developing breast cancer, but who arenot know to have cancer. In most individuals, ducts (D1-D4) from bothbreasts were serially sampled at 6-9 month intervals (left to right,respectively). Shown is the cumulative methylation of RARβ, RASSF1A,TWIST, Cyclin D2, HIN1, ESR1, APC1, BRCA1, BRCA2, and p16 (CDKN2A) geneswithin the DNA sample. The relative level of methylation of each gene isindicated by the height of the bar. Total possible units=1000 (10genes×100%).

FIGS. 13A-B are bar graphs showing the level of gene promoterhypermethylation in cells derived from nipple aspiration fluid (n=10)from a series of patients (n=7) at high risk of developing breastcancer, but who are not know to have cancer. FIG. 13A shows thecumulative methylation of RARβ, RASSF1A, TWIST, Cyclin D2, HIN1, ESR1,APC1, BRCA1, and BRCA2 genes within the DNA sample. The relative levelof methylation of each gene is indicated by the height of the bar. Totalpossible units=900 (9 genes×100%). FIG. 13B shows high levels of HIC1methylation were discovered these cytologically benign samples, asindicated by the bar graph showing % methylation within each sample.

DETAILED DESCRIPTION OF THE INVENTION

In the invention methods employing a two-step quantitativemultiplex-methylation specific PCR (QM-MSP), many genes can beco-amplified from amounts of sample previously used for just one geneand a combined methylation score can be generated for the panel ofgenes. The QM-MSP technique combines the sensitivity of multiplex PCRwith the quantitative features of quantitative methylation-specific PCR(Q-MSP) in such a way that a panel of genes whose hypermethylation isassociated with a type of carcinoma can be co-amplified from limitingamounts of DNA derived from tissue or samples sources of the subjectbeing tested. The invention methods also provide quantitative definitionof the extent of gene hypermethylation in normal appearing tissues on agene-by-gene basis. Thus, the invention methods may be used to morepowerfully discriminate between normal or benign tissues and malignanttissues and to monitor or assess the course of cancer development in asubject.

The invention methods are also broadly applicable to evaluation of anyof hundreds of genes from hypermethylated regions of genomic DNA derivedfrom but not limited to human or non-human mammals, plants and insects.For example in mammals, such as humans, samples may be derived fromdifferent tissue sources and bodily fluids, including one or moreselected from tumor-associated tissue, normal tissue, blood, serum,plasma, ductal lavage fluid, nipple aspiration fluid, lymph, duct cells,lymph nodes, and bone marrow of the subject being tested. In mammals,conditions associated with aberrant methylation of genes that can bedetected or monitored include, but are not limited to, carcinomas andsarcomas of all kinds, including one or more specific types of cancer,e.g., breast cancer, an alimentary or gastrointestinal tract cancer suchas colon, esophageal and pancreatic cancer, a liver cancer, a skincancer, an ovarian cancer, an endometrial cancer, a prostate cancer, alymphoma, hematopoietic tumors, such as a leukemia, a kidney cancer, alung cancer, a bronchial cancer, a muscle cancer, a bone cancer, abladder cancer or a brain cancer, such as astrocytoma, anaplasticastrocytoma, glioblastoma, medulloblastoma, and neuroblastoma and theirmetastases. Suitable pre-malignant lesions to be detected or monitoredusing the invention include, but are not limited to, lobular carcinomain situ and ductal carcinoma in situ. The invention methods can be usedto assay the DNA of any mammalian subject, including humans, pet (e.g.,dogs, cats, ferrets) and farm animals (meat and dairy), and race horses.

Samples obtained from other multicellular organisms, such as insects andplants, may also be evaluated for gene methylation status using theinvention methods. For example, the methylation status of genes mayserve as an indicator of heritability and flexibility of epigeneticstates in such subjects. Thus, gene methylation is linked to acquisitionof different characteristics in the organism (Frank Lyko Trends in Gen(2001) 17:169-172, Finnegan E. J. et al. Annu Rev Plant Physiol PlantMol Biol (1998) 49:223-247).

In one embodiment, the invention provides methods for determining themethylation status of a DNA sample by co-amplifying a plurality of DNAsequences using a mixture of DNA sequence-specific, methylationstatus-independent outer primer pairs that selectively hybridize totheir cognate DNA sequences under conditions that allow generation of afirst amplification product containing first amplicons. Real time PCR isthen used to amplify unmethylated and/or methylated DNA from the firstamplicons under conditions that allow generation of second amplificationproducts for one or more genes, using for a gene two sets of primerscomprising a DNA sequence-specific, methylation status-dependent(unmethylated or methylated) inner primer pair and a (unmethylated ormethylated) DNA sequence-specific probe. Second amplicons are detectedby using one or more distinguishable optically detectable labels perreaction. A combination of inner primer pair and probe selectivelyhybridizes to one first amplicon, and the sets of inner primer pairs andprobes collectively hybridize to a plurality of first amplicons in thefirst amplification product. Signal intensities of the one or moredistinguishable labels in the second amplification products are detectedto determine the amount of methylation in a gene. A combined methylationvalue is derived for the methylation in the DNA sequences in the testsample from amounts of methylation determined for the secondamplification products in a panel of genes, as compared with thecombined methylation value in comparable normal DNA samples to determinethe methylation status of the DNA sample.

In yet another embodiment, the invention provides methods of diagnosingdevelopment of a condition such as pregnancy, preeclampsia, or eclampsiaassociated with aberrant methylation of DNA in tissue of a subject. Inthis embodiment, the invention methods comprise co-amplifying aplurality of DNA sequences using a mixture of DNA sequence-specific,methylation status-independent outer primer pairs that selectivelyhybridize to their cognate DNA sequences under conditions that allowgeneration of a first amplification product containing first amplicons.Real time PCR is then used to amplify unmethylated and/or methylated DNAfrom the first amplicons under conditions that allow generation ofsecond amplification products for one or more genes, using for a genetwo sets of primers, comprising a DNA sequence-specific, methylationstatus-dependent (unmethylated or methylated) inner primer pair and a(unmethylated or methylated) DNA sequence-specific probe. Secondamplicons are detected by using one or more distinguishable opticallydetectable labels per reaction. A combination of inner primer pair andprobe selectively hybridizes to one first amplicon, and the sets ofinner primer pairs and probes collectively hybridize to a plurality offirst amplicons in the first amplification product. Signal intensitiesof the one or more distinguishable labels in the second amplificationproducts are detected to determine the amount of methylation in a gene.A combined methylation value is derived for the methylation in the DNAsequences in the test sample from amounts of methylation determined forthe second amplification products across a panel of genes as comparedwith a combined methylation value in comparable normal tissues todiagnose the state of development of the condition in the subject.

In still another embodiment, the invention provides methods fordiagnosing development of a carcinoma associated with hypermethylationof CpG island DNA in carcinoma-associated tissue of a subject byco-amplifying CpG islands in a subject sample comprising severaldifferent DNA sequences isolated from the carcinoma-associated tissueusing a mixture of DNA sequence-specific, methylation status-independentouter primer pairs that selectively hybridize to one or more of the DNAsequences under conditions that allow generation of a firstamplification product containing first amplicons. Then, real time PCR isused to co-amplify the CpG islands in the first amplicons underconditions that allow generation of one or more second amplificationproducts, using one or more members of a set of DNA sequence-specificprobes comprising distinguishable fluorescent moieties and one or moremembers of a set of DNA sequence-specific methylation status-dependentinner primer pairs that hybridize to one first amplicon, wherein thesets of probes and inner primer pairs collectively hybridize to aplurality of different first amplicons in the first amplificationproduct. Fluorescence due to the presence of distinguishable fluorescentmoieties in the second amplification products is detected to determinethe amount of methylation of the CpG islands therein. The combinedamount of hypermethylation in CpG islands in the tumor-related tissuecompared with a combined amount of methylation in comparable normaltissue is evaluated to diagnose the state of development of the cancerin the subject. For example, the sum of the extent (e.g. percentage) ofmethylation of the second amplification products across a panel of genescan be used to measure a cumulative amount of hypermethylation in CpGislands in the tumor-related tissue as compared with a cumulative amountof methylation in comparable normal tissue to diagnose the state ofdevelopment of the carcinoma in the subject.

In yet another embodiment, the invention methods can be used fordiagnosing the state of development of a condition, such as any cancerand metastases thereof, associated with hypermethylation or aberrantmethylation of a gene in a mammalian subject by co-amplifying aplurality of different DNA sequences isolated from tissue of the subjectassociated with the condition co-amplifying a plurality of DNA sequencesusing a mixture of DNA sequence-specific, methylation status-independentouter primer pairs that selectively hybridize to their cognate DNAsequences under conditions that allow generation of a firstamplification product containing first amplicons. Real time PCR is thenused to amplify unmethylated and/or methylated DNA from the firstamplicons under conditions that allow generation of second amplificationproducts for one or more genes, using for a gene two sets of primerscomprising a DNA sequence-specific, methylation status-dependent(unmethylated or methylated) inner primer pair and a (unmethylated ormethylated) DNA sequence-specific probe. Second amplicons are detectedby using one or more distinguishable optically detectable labels perreaction. A combination of inner primer pair and probe selectivelyhybridizes to one first amplicon, and the sets of inner primer pairs andprobes collectively hybridize to a plurality of first amplicons in thefirst amplification product. Signal intensities of the one or moredistinguishable labels in the second amplification products are detectedto determine the amount of methylation in a gene. A combined methylationvalue is derived for the methylation in the DNA sequences in the testsample from amounts of methylation determined for the secondamplification products in a panel of genes, as compared with thecombined methylation value in comparable normal tissue sample toindicate the state of development of the condition in the subject.

Optionally, in any of the invention methods, a single combination ofinner primer pair and probe may be used for amplification using realtime PCR in separate aliquots of the first amplification product. In anycase, the sets of probes and inner primer pairs collectively amplify aplurality of different first amplicons in the first amplificationproduct.

In another embodiment, the invention provides kits for determining themethylation status of a plurality of DNA sequences in a DNA sample. Theinvention kits include a set of DNA sequence-specific, methylationstatus-independent outer primer pairs that selectively hybridize to oneor more of the plurality of DNA sequences under conditions that allowgeneration of a first amplification product containing first amplicons,a set of DNA sequence-specific, methylation status-dependent innerprimer pairs, and a set of DNA sequence-specific probes with one or moredistinguishable optically detectable labels. A combination of innerprimer pair and probe selectively hybridizes to one or more firstamplicon, and the sets of inner primer pairs and probes collectivelyhybridize to a plurality of first amplicons in the first amplificationproduct. The invention kits may include two sets of the set of DNAsequence-specific, methylation status-dependent inner primer pairs, asubset that specifically hybridizes to methylated first amplicons and asubset that specifically hybridizes to unmethylated first amplicons.Illustrative primers are exemplified in the Examples herein. In certainembodiments, the kit may further provide a set of instructions forperforming the invention methods using the contents of the kit.

The invention methods combine the principles of MSP, multiplex MSP,quantitative real-time PCR (Q-PCR) with quantitative MSP (Q-MSP) in aprocedure referred to herein as quantitative multiplexmethylation-specific PCR (QM-MSP) (FIGS. 1 and 2). The studies describedherein have shown that this combination of procedures can detect as fewas 1-10 methylated copies of DNA in a mixture of about 100,000 copies ofunmethylated DNA (Table 3 and FIG. 3) and 40 pg of methylated genomicDNA in up to 1500-fold excess unmethylated DNA (FIG. 4). This outcomecompares favorably to Q-MSP with a sensitively of 1:10,000 (Trinh B. N.et al., supra) and conventional MSP with a sensitivity of 1:1000 (HermanJ. G. et al., supra). In addition, reactions are specific since nocross-reactivity was observed between methylated and unmethylatedprimers even in mixtures consisting of more than 10⁵-fold excess of oneor the other DNA (Table 3, FIG. 3). Also, it has been demonstrated thatusing the level of unmethylated product for each gene as the internalcontrol for assessing the extent of methylated gene product presentprovides accurate quantitation (Table 4).

Quantitative Real-Time PCR (Q-PCR)

The ability to monitor the real-time progress of the PCR changes the wayone approaches PCR-based quantification of DNA and RNA. Reactions arecharacterized by the point in time during cycling when amplification ofa PCR product is first detected rather than the amount of PCR productaccumulated after a fixed number of cycles. The higher the starting copynumber of the nucleic acid target, the sooner a significant increase influorescence is observed. FIGS. 3 and 4 show representativeamplification plots. An amplification plot is the plot of fluorescencesignal versus cycle number. In the initial cycles of PCR, there islittle change in fluorescence signal. This defines the baseline for theamplification plot. An increase in fluorescence above the baselineindicates the detection of accumulated PCR product. A fixed fluorescencethreshold can be set above the baseline. The parameter C_(T) (thresholdcycle) is defined as the fractional cycle number at which thefluorescence passes the fixed threshold. For example, the PCR cyclenumber at which fluorescence reaches a threshold value of 10 times thestandard deviation of baseline emission may be used as C_(T) and it isinversely proportional to the starting amount of target cDNA. A plot ofthe log of initial target copy number for a set of standards versusC_(T) is a straight line. Quantification of the amount of target inunknown samples is accomplished by measuring C_(T) and using thestandard curve to determine starting copy number.

The entire process of calculating C_(T)S, preparing a standard curve,and determining starting copy number for unknowns can be performed bysoftware, for example that of the 7700 system or 7900 system of AppliedBiosystems. Real-time PCR requires an instrumentation platform thatconsists of a thermal cycler, computer, optics for fluorescenceexcitation and emission collection, and data acquisition and analysissoftware. These machines, available from several manufacturers, differin sample capacity (some are 96-well standard format, others processfewer samples or require specialized glass capillary tubes), method ofexcitation (some use lasers, others broad spectrum light sources withtunable filters), and overall sensitivity. There are alsoplatform-specific differences in how the software processes data.Real-time PCR machines are available at core facilities or labs thathave the need for high throughput quantitative analysis.

Briefly, in the Q-PCR method the number of target gene copies can beextrapolated from a standard curve equation using the absolutequantitation method. For each gene, cDNA from a positive control isfirst generated from RNA by the reverse transcription reaction. Usingabout 1 μl of this cDNA, the gene under investigation is amplified usingthe primers by means of a standard PCR reaction. The amount of ampliconobtained is then quantified by spectrophotometry and the number ofcopies calculated on the basis of the molecular weight of eachindividual gene amplicon. Serial dilutions of this amplicon are testedwith the Q-PCR assay to generate the gene specific standard curve.Optimal standard curves are based on PCR amplification efficiency from90 to 100% (100% meaning that the amount of template is doubled aftereach cycle), as demonstrated by the slope of the standard curveequation. Linear regression analysis of all standard curves should showa high correlation (R² coefficient≧0.98). Genomic DNA can be similarlyquantified.

When measuring transcripts of a target gene, the starting material,transcripts of a housekeeping gene are quantified as an endogenouscontrol. Beta-actin is one of the most used nonspecific housekeepinggenes. For each experimental sample, the value of both the target andthe housekeeping gene are extrapolated from the respective standardcurve. The target value is then divided by the endogenous referencevalue to obtain a normalized target value independent of the amount ofstarting material.

Primer and Probe Design for QM-MSP

In practice of the invention methods, the above-described quantitativereal-time PCR methodology has been adapted to perform quantitativemethylation-specific PCR (QM-MSP) by utilizing the external primerspairs in round one (multiplex) PCR and internal primer pairs in roundtwo (real time MSP) PCR. Thus each set of genes has one pair of externalprimers and two sets of three internal primers/probe (internal sets arespecific for unmethylated or methylated DNA). The external primer pairscan co-amplify a cocktail of genes, each pair selectively hybridizing toa member of the panel of genes being investigated using the inventionmethod. Primer pairs are designed to exclude CG dinucleotides, therebyrendering DNA amplification independent of the methylation status of thepromoter DNA sequence. Therefore methylated and unmethylated DNAsequences internal to the binding sites of the external primers areco-amplified for any given gene by a single set of external primersspecific for that gene. The external primer pair for a gene beinginvestigated is complementary to the sequences flanking the CpG islandthat is to be queried in the second round of QM-MSP. For example, thesequences of external primers set forth in Table 1 below are used formultiplex PCR (first round PCR) of genes associated with primary breastcancer (Fackler M. J. et al, Int. J Cancer (2003) 107:970-975; FacklerM. J. et al. Cancer Res (2004) 64:4442-4452).

Internal PCR primers used for quantitative real-time PCR of methylatedand unmethylated DNA sequences (round two QM-MSP) are designed toselectively hybridize to the first amplicon produced by the first roundof PCR for one or more members of the panel of DNA sequences beinginvestigated using the invention method and to detect the methylationstatus, i.e., whether methylated (M) or unmethylated (U), of the CpGislands in the first amplicons to which they bind. Thus for each memberof the starting panel of promoter DNA sequences used in an inventionassay, separate QM-MSP reactions are conducted to amplify the firstamplicon produced in the first round of PCR using the respectivemethylation-specific primer pair and using the respectiveunmethylated-specific primer pair.

In round two of QM-MSP a single gene or a cocktail of two or more genescan be co-amplified using distinguishable fluorescence labeled probes.The probes used in the round two QM-MSP of the invention method aredesigned to selectively hybridize to a segment of the first ampliconlying between the binding sites of the respective methylation-statusspecific internal PCR primer pair. Polynucleotide probes suitable foruse in real-time PCR include, but are not limited to, a bilabeledoligonucleotide probe, such as a molecular beacon or a TaqMan™ probe,which include a fluorescent moiety and a quencher moiety. In a molecularbeacon the fluorescence is generated due to hybridization of the probe,which displaces the quencher moiety from proximity of the fluorescentmoiety due to disruption of a stem-loop structure of the bilabeledoligonucleotide. Molecular beacons, such as Amplifluor™ or TriStar™reagents and methods are commercially available (Stratagene; Intergen).In a TaqMan™ probe, the fluorescence is progressively generated due toprogressive degradation of the probe by Taq DNA polymerase during roundsof amplification, which displaces the quencher moiety from thefluorescent moiety. Once amplification occurs, the probe is degraded bythe 5′-3′ exonuclease activity of the Taq DNA polymerase, and thefluorescence can be detected, for example by means of a laser integratedin the sequence detector. The fluorescence intensity, therefore, isproportional to the amount of amplification product produced.

In one embodiment, fluorescence from the probe is detected and measuredduring a linear amplification reaction and, therefore, can be used todetect generation of the linear amplification product.

Amplicons in the 80-150 base pair range are generally favored becausethey promote high-efficiency assays that work the first time. Inaddition, high efficiency assays enable quantitation to be performedusing the absolute quantitation method. Quantitation of the copy numberof unmethylated (U) and methylated (M) DNA amplicons for a specific geneeliminates the need to use actin as an estimate of DNA input, since U+Mis taken to approximate the total number of copies of DNA amplicons forany given gene in the first amplicon product (derived from round one,multiplex PCR of QM-MSP).

Whenever possible, primers and probes can be selected in a region with aG/C content of 20-80%. Regions with G/C content in excess of this maynot denature well during thermal cycling, leading to a less efficientreaction. In addition, G/C-rich sequences are susceptible tonon-specific interactions that may reduce reaction efficiency. For thisreason, primer and probe sequences containing runs of four or more Gbases are generally avoided. A/T-rich sequences require longer primerand probe sequences in order to obtain the recommended TMs. This israrely a problem for quantitative assays; however, TaqMan™ probesapproaching 40 base pairs can exhibit less efficient quenching andproduce lower synthesis yields.

For example, external primer pairs, internal primer pairs andgene-specific probes for determining the methylation and unmethylationstatus of certain genes associated with primary breast cancer, RASSF1A,TWIST, Cyclin D2, and HIN1, are set forth in Tables 1 and 2 below.

TABLE 1 Sequence of Multiplex External Primers(Used in Round 1 PCR of QM-MSP) Primer Name Primer Sequence OrientationSEQ ID NO. Cyclin D2 Ext F Tattttttgtaaagatagttttgat ForwardSEQ ID NO: 1 Cyclin D2 Ext R Tacaactttctaaaaaataaccc ReverseSEQ ID NO: 2 RASSF1A Ext F(2) Gttttatagtttttgtatttagg ForwardSEQ ID NO: 3 RASSF1A Ext R(2) Aactcaataaactcaaactccc ReverseSEQ ID NO: 4 TWIST Ext R(4) Cctcccaaaccattcaaaaac Forward SEQ ID NO: 5TWIST Ext F(3) Gagatgagatattatttattgtg Reverse SEQ ID NO: 6 RARB Ext FGtaggagggtttattttttgtt Forward SEQ ID NO: 7 RARB Ext R(2)Aattacattttccaaacttactc Reverse SEQ ID NO: 8 HIN1 Ext F(2)Gtttgttaagaggaagtttt Forward SEQ ID NO: 9 HIN1 Ext RCcgaaacatacaaaacaaaaccac Reverse SEQ ID NO: 10 ACTB Ext FTatataggttggggaagtttg Forward SEQ ID NO: 11 ACTB Ext RTataaaaacataaaacctataacc Reverse SEQ ID NO: 12 ESR1 Ext F(2)Ggtgtatttggatagtagtaag Forward SEQ ID NO: 46 ESR1 Ext R(3)Ctccaaataataaaacacctact Reverse SEQ ID NO: 47 APC1 Ext F(2)Aaaaccctataccccactac Forward SEQ ID NO: 48 APC1 Ext R(2)Ggttgtattaatatagttatatgt Reverse SEQ ID NO: 49 BRCA1 Ext FTattttgagaggttgttgtttag Forward SEQ ID NO: 50 BRCA1 Ext RAaacatcacttaaaccccctat Reverse SEQ ID NO: 51 BRCA2 Ext FGttgggatgtttgataaggaat Forward SEQ ID NO: 52 BRCA2 Ext RAtcacaaatctatcccctcac Reverse SEQ ID NO: 53 P16 Ext F(3)Aaagaggaggggttggttg Forward SEQ ID NO: 54 P16 Ext R(5)Aaccctctacccacctaaat Reverse SEQ ID NO: 55 HIC1 Ext FTttagttgagggaaggggaa Forward SEQ ID NO: 56 HIC1 Ext RAactacaacaacaactacctaa Reverse SEQ ID NO: 57

TABLE 2 Sequences of QM-MSP Primers (Used in Round 2 PCR of QM-MSP)QM-MSP Primer Primer Name Sequences Orientation Status Cyclin D2 RT-tttgatttaaggatgcgttagagtacg SEQ ID NO: 13 Forward M FM Cyclin D2 RT-actttctccctaaaaaccgactacg SEQ ID NO: 14 Reverse M RM Cyclin D2 Maatccgccaacacgatcgacccta SEQ ID NO: 15 Reverse M Probe Cyclin D2 RT-ttaaggatgtgttagagtatgtg SEQ ID NO: 16 Forward U FUM Cyclin D2 RT-aaactttctccctaaaaaccaactacaat SEQ ID NO: 17 Reverse U RUM Cyclin D2 UMaatccaccaacacaatcaaccctaac SEQ ID NO: 18 Reverse U Probe RASSF1A RT-gcgttgaagteggggttc SEQ ID NO: 19 Forward M FM RASSF1A RT-cccgtacttcgctaactttaaacg SEQ ID NO: 20 Reverse M RM RASSF1A Macaaacgcgaaccgaacgaaacca SEQ ID NO: 21 Reverse M Probe RASSF1A RT-ggtgttgaagttggggtttg SEQ ID NO: 22 Forward U FUM RASSF1A RT-cccatacttcactaactttaaac SEQ ID NO: 23 Reverse U RUM RASSF1A UMctaacaaacacaaaccaaacaaaacca SEQ ID NO: 24 Reverse U Probe TWIST RT-FMgttagggttcgggggcgttgtt SEQ ID NO: 25 Forward M TWIST RT-RMccgtcgccttcctccgacgaa SEQ ID NO: 26 Reverse M TWIST M-aaacgatttccttccccgccgaaa SEQ ID NO: 27 Reverse M Probe TWIST RT-ggtttgggggtgttgtttgtatg SEQ ID NO: 28 Forward U FUM(3) TWIST-RT-cccacctcctaaccaccctcc SEQ ID NO: 29 Reverse U RUM (3) TWIST UMaaacaatttccttccccaccaaaaca SEQ ID NO: 30 Reverse U Probe RARB RT-FMagaacgcgagcgattcgagtag SEQ ID NO: 31 Forward M RARB RT-RMtacaaaaaaccttccgaatacgtt SEQ ID NO: 32 Reverse M RARB M Probeatcctaccccgacgatacccaaac SEQ ID NO: 33 Reverse M RARB RT-ttgagaatgtgagtgatttgagtag SEQ ID NO: 34 Forward U FUM RARB RT-ttacaaaaaaccttccaaatacattc SEQ ID NO: 35 Reverse U RUM RARB UMaaatcctaccccaacaatacccaaac SEQ ID NO: 36 Reverse U Probe HIN1 RT-FMtagggaagggggtacgggttt SEQ ID NO: 37 Forward M HIN1 RT-RMcgctcacgaccgtaccctaa SEQ ID NO: 38 Reverse M HIN1 M Probeacttcctactacgaccgacgaacc SEQ ID NO: 39 Reverse M HIN1-RT-FUMaagtttttgaggtttgggtaggga SEQ ID NO: 40 Forward U (2) HIN1 RT-RUMaccaacctcacccacactccta SEQ ID NO: 41 Reverse U (2) HIN1 UMcaacttcctactacaaccaacaaacc SEQ ID NO: 42 Reverse U Probe ACTB Ftggtgatggaggaggtttagtaagt SEQ ID NO: 43 Forward Indep ACTB Raaccaataaaacctactcctcccttaa SEQ ID NO: 44 Reverse Indep ACTB Probeaccaccacccaacacacaataacaaacaca SEQ ID NO: 45 Reverse Indep ESR1 RT-FUMtgttgtgtataattattttgagggt SEQ ID NO: 58 Forward M ESR1 RT-RUMccaatctaaccataaacctacaca SEQ ID NO: 59 Reverse M ESR1 UMcaacaaccacaacattaaactcataaa SEQ ID NO: 60 Reverse M Probe ESR1 RT-FMcgtcgtgtataattatttcgagg SEQ ID NO: 61 Forward U ESR1 RT-RMgatctaaccgtaaacctacgcg SEQ ID NO: 62 Reverse U ESR1 M Probecgacgaccgcgacgttaaactcgt SEQ ID NO: 63 Reverse U APC1 RT-FUMtaaatacaaaccaaaacactccc SEQ ID NO: 64 Forward M APC1 RT-RUMgttatatgttggttatgtgtgttt SEQ ID NO: 65 Reverse M APC1 UMttcccatcaaaaacccaccaattaac SEQ ID NO: 66 Reverse M probe APC1 RT-FMaatacgaaccaaaacgctccc SEQ ID NO: 67 Forward U APC1 RT-RMtatgtcggttacgtgcgtttatat SEQ ID NO: 68 Reverse U APC1 M Probecccgtcgaaaacccgccgatta SEQ ID NO: 69 Reverse U BRCA1 RT-tggtaatggaaaagtgtgggaa SEQ ID NO: 70 Forward M FUM BRCA1 RT-cccatccaaaaaatctcaacaaa SEQ ID NO: 71 Reverse M RUM(4) BRCA1 UMctcacaccacacaatcacaattttaat SEQ ID NO: 72 Reverse M probe BRCA1 RT-FMtttcgtggtaacggaaaagcg SEQ ID NO: 73 Forward U BRCA1 RT-ccgtccaaaaaatctcaacgaa SEQ ID NO: 74 Reverse U RM BRCA1 Mctcacgccgcgcaatcgcaattt SEQ ID NO: 75 Reverse U Probe BRCA2 RT-atttttgggtggtgtgtgtgtt SEQ ID NO: 76 Forward M FUM BRCA2 RT-tcaaaaactcacaccacaaacc SEQ ID NO: 77 Reverse M RUM BRCA2 UMaaccacataacaccataacacaacac SEQ ID NO: 78 Reverse M Probe BRCA2 RT-FMtttgattttcgggtggtgcgt SEQ ID NO: 79 Forward U BRCA2 RT-tcaaaaactcgcgccacaaac SEQ ID NO: 80 Reverse U RM BRCA2 Maaccacgtaacgccgtaacgcga SEQ ID NO: 81 Reverse U Probe P16 RT-ttattagagggtggggtggattgt SEQ ID NO: 82 Forward M FUM(2) P16 RT-RUMcaaccccaaaccacaaccataa SEQ ID NO: 83 Reverse M P16 UM Probectactccccaccacccactacct SEQ ID NO: 84 Reverse M P16 RT-FM(2)ttattagagggtggggcggatcgc SEQ ID NO: 85 Forward U P16 RT-RMgaccccgaaccgcgaccgtaa SEQ ID NO: 86 Reverse U P16 M Probeagtagtatggagtcggcggcggg SEQ ID NO: 87 Reverse U HIC1 RT-FUMgggttaggtggffagggtgtt SEQ ID NO: 88 Forward M HIC1 RT-RUMtaaccaaacacctccatcatatc SEQ ID NO: 89 Reverse M HIC1 UMaaacacacaccaaccaaataaaaaccat SEQ ID NO: 90 Reverse M Probe HIC1 RT-FMggttaggcggttagggcgtc SEQ ID NO: 91 Forward U HIC1 RT-RMccgaacgcctccatcgtatc SEQ ID NO: 92 Reverse U HIC1 M Probecacacaccgaccgaataaaaaccgt SEQ ID NO: 93 Reverse U

The TaqMan™ probe used in the Example herein contains both a fluorescentreporter dye at the 5′ end, such as 6-carboxyfluorescein (6-FAM:emission λ_(max)=518 nm) and a quencher dye at the 3′ end, such as6-carboxytetramethylrhodamine, (TAMRA; emission λ_(max)=582 nm). Thequencher can quench the reporter fluorescence when the two dyes areclose to each other, which happens in an intact probe. Other reporterdyes include but are not limited to VIC™ and TET™ and these can be usedin conjunction with 6-FAM to co-amplify genes by quantitative real timePCR. For instance in round two QM-MSP, unmethylated (using a 6-FAM/TAMRAprobe) and unmethylated RARE (using a VIC/TAMRA probe) either can beco-amplified (FIG. 1) or can be assayed as single genes.

Thermal Cycling Parameters

The round two QM-MSP reactions are designed to be run as single genereactions or in multiplex using automated equipment, as are other typesof real time PCR. Thermal cycling parameters useful for performing realtime PCR are well known in the art and are illustrated in the Examplesherein. In certain embodiments, quantitative assays can be run using thesame universal thermal cycling parameters for each assay. Thiseliminates any optimization of the thermal cycling parameters and meansthat multiple assays can be run on the same plate without sacrificingperformance. This benefit allows combining two or more assays into amultiplex assay system, in which the option to run the assays underdifferent thermal cycling parameters is not available.

Using the QM-MSP approach, it is possible to compile gene panels thatare designed to address specific questions, or to provide intermediatemarkers or endpoints for clinical protocols. For example, when usingde-methylating agents, a panel can be designed to query pathway-specificgenes for their use as intermediate markers in specific trials ofchemopreventive agents (Fackler M. J. et al. J Mammary Gland BiolNeoplasia (2003) 8:75-89).

In summary, the invention methods can be used to assess the methylationstatus of multiple genes, using only picograms of DNA. A cumulativescore of hypermethylation among multiple genes better distinguishesnormal or benign from malignant tissues. While the studies describedherein show that some normal or benign tissue may have low-level genepromoter hypermethylation, it is also shown that distinct differencesexist between the levels of normal and malignant tissues. Using QM-MSP,therefore, it is possible to objectively define the range ofnormal/abnormal DNA sequence hypermethylation in a manner that istranslatable to a larger clinical setting. The invention methods mayalso be used to examine cumulative hypermethylation in benign conditionsand as a predictor of conditions, such as various cancers and theirmetastases that are associated with DNA hypermethylation. The inventionmethod may also predict conditions not associated with cancers, such aspre-eclampsia or eclampsia (Hueller H. M. et al. M Clin Chem (2004)50:1065-1068).

In one embodiment, the primer extension reaction is a linearamplification reaction, wherein the primer extension reaction isperformed over a number of cycles, and the linear amplification productthat is generated is detected.

Due to the sensitivity of the invention methods, the first amplificationproduct is optionally diluted from about 1:5 to about 1:10⁵ andoptionally separate aliquots of the first amplification product arefurther subjected to QM-MSP as described herein.

In real-time PCR, as used in the invention methods, one or more aliquots(usually dilute) of the first amplification product is amplified with atleast a first primer of an internal amplification primer pair, which canselectively hybridize to one or more amplicon in the first amplificationproduct, under conditions that, in the presence of a second primer ofthe internal amplification primer pair, and a fluorescent probe allowsthe generation of a second amplification product. Detection offluorescence from the second amplification product(s) provides a meansfor real-time detection of the generation of a second amplificationproduct and for calculation of the amount of gene specific secondamplification product produced. Alternatively, the first amplificationproducts used for the real time PCR reaction(s) may be contacted withboth 1) a combination of a probe and DNA sequence-specific internalprimer pair that recognizes only a methylated CpG island in the firstamplicon and 2) a combination of a probe and DNA sequence-specificinternal primer pair that recognizes an unmethylated CpG island for eachof the DNA sequences in the panel of DNA sequences.

Thus, in one embodiment, the real-time PCR amplification in an inventiontwo-step assay is, in fact, a group of real-time PCR reactions, whichmay be conducted together or using two separate aliquots of the firstamplification product, for each of the first amplicons (i.e., for eachDNA sequence in the panel that were selected for the assay). In thisembodiment, determination of the methylation status of a DNA sequence,such as one containing a CpG island, employs both amethylation-determining and an unmethylation determining internal primerpair for each amplicon contained in the first amplification product, oneto determine if the gene is unmethylated and one to determine if thegene is methylated. The real time PCR reactions in the secondamplification step of the invention methods can conveniently beconducted sequentially or simultaneously in multiplex. Separate, usuallydilute, aliquots of the first amplification reaction may be used foreach of the two methylation status determining reactions. For example,the reactions can conveniently be performed in the wells of a 96 or 384microtiter plate. For convenience, the methylated and unmethylatedstatus determining second reactions for a target gene may be conductedin adjacent wells of a microtiter plate for high throughput screening.Alternatively, several genes, for example 2 to 5 genes, may besimultaneously amplified in a single real time PCR reaction if theprobes used for each first amplicon are distinguishably labeled.

For example, two to five distinguishable fluorescence signals from thesecond amplification product(s) may be accumulated to determine thecumulative incidence or level of methylation of the DNA sequences,especially of CpG islands therein, in the several genes included in theassay. These cumulative results are compared with the cumulative resultssimilarly obtained by conducting the two step QM-MSP assay on comparableDNA sequences (e.g., promoter DNA sequences) obtained from comparablenormal tissue of the same type or types as used in the assay.

Any of the known methods for conducting cumulative or quantitative “realtime PCR” may be used in the second amplification step so long as thefirst amplicons in the first amplification product are contacted withone or more members of a set of polynucleotide probes that are labeledwith distinguishable optically detectable labels, one or more members ofthe set being designed to selectively hybridize to one or more of theDNA sequences being tested, while the set cumulatively binds to thevarious DNA segments being tested contained in the first amplicons ofthe first amplification product. In addition, the first amplicons mayalso be contacted with such a set of probes and one or more members of aset of DNA sequence-specific methylation status-dependent inner primerpairs, wherein the set of inner primer pairs collectively bind to thevarious first amplicons in the first amplification product. In round twoQM-MSP, additional genes can be co-amplified provided that each geneprimer set incorporates a different color fluorescent probe.

It should be recognized that an amplification “primer pair” as the termis used herein requires what are commonly referred to as a forwardprimer and a reverse primer, which are selected using methods that arewell known and routine and as described herein such that anamplification product can be generated therefrom.

As used herein, the phrase “conditions that allow generation of anamplification product” or of “conditions that allow generation of alinear amplification product” means that a sample in which theamplification reaction is being performed contains the necessarycomponents for the amplification reaction to occur. Examples of suchconditions are provided in Example 1 and include, for example,appropriate buffer capacity and pH, salt concentration, metal ionconcentration if necessary for the particular polymerase, appropriatetemperatures that allow for selective hybridization of the primer orprimer pair to the template nucleic acid molecule, as well asappropriate cycling of temperatures that permit polymerase activity andmelting of a primer or primer extension or amplification product fromthe template or, where relevant, from forming a secondary structure suchas a stem-loop structure. Such conditions and methods for selecting suchconditions are routine and well known in the art (see, for example,Innis et al., “PCR Strategies” (Academic Press 1995); Ausubel et al.,“Short Protocols in Molecular Biology” 4th Edition (John Wiley and Sons,1999); “A novel method for real time quantitative RT-PCR” Gibson U. E.et al. Genome Res (1996) 6:995-1001; “Real time quantitative PCR” HeidC. A. et al. Genome Res (1996) 6:986-994).

As used herein, the term “selective hybridization” or “selectivelyhybridize” refers to hybridization under moderately stringent or highlystringent conditions such that a nucleotide sequence associates with aselected nucleotide sequence but not with unrelated nucleotidesequences. Generally, an oligonucleotide useful as a probe or primerthat selectively hybridizes to a selected nucleotide sequence is atleast about 15 nucleotides in length, usually at least about 18nucleotides, and particularly about 21 nucleotides in length or more inlength. Conditions that allow for selective hybridization can bedetermined empirically, or can be estimated based, for example, on therelative GC:AT content of the hybridizing oligonucleotide and thesequence to which it is to hybridize, the length of the hybridizingoligonucleotide, and the number, if any, of mismatches between theoligonucleotide and sequence to which it is to hybridize (see, forexample, Sambrook et al., “Molecular Cloning: A laboratory manual” (ColdSpring Harbor Laboratory Press 1989)).

The phrase “comparable normal tissue” as used herein means thathistologically apparently normal tissue(s) are collected from tissuesite(s) in the subject adjacent to the site(s) from which the tumortissue is collected. Alternatively, the “comparable normal tissue” canbe collected from sites(s) of unrelated healthy donors that anatomicallyrelated to the site(s) of subject tumor tissue used (e.g., same tissuetype), or the results representing the accumulated incidence of thetumor marker in anatomically related apparently normal tissue site(s) ofnormal subjects can be compared with the results of the assay obtainedfrom the subject's own tumor-associated tissue. Similarly, the phrase “acomparable normal DNA sample” as used herein means that the plurality ofgenomic DNA sequences that is being tested for methylation status, suchas in a plant or insect, is matched with a panel of genomic. DNAsequences of the same genes from a “normal” organism of the samespecies, family, and the like, for comparison purposes. For example, asubstantial cumulative increase or decrease in the methylation level inthe test sample as compared with the normal sample (e.g., the cumulativeincidence of the tumor marker in a test DNA panel compared with thatcumulatively found in comparable apparently normal tissue) is a reliableindicator of the presence of the condition being assayed.

The invention methods can be used to assay the DNA of any mammaliansubject, including humans, pets (e.g., dogs, cats, ferrets) and farmanimals (meat and dairy), and race horses. The invention methods canalso be used to assay the DNA of plants (“DNA methylation in plants”Finnegan E. J. et al. Annu Rev Plant Physiol Plant Mol Biol (1998)49:223-247) and insects (“DNA methylation learns to fly” Frank LykoTrends in Gen (2001) 17:169-172).

The invention methods are illustrated using primary breast cancer as anexample (Fackler M. J. et al, Int. J. Cancer (2003) 107:970-975; FacklerM. J. et al. Cancer Res (2004) 64:4442-4452). In breast cancer, samplescan be collected from such tissue sources as ductal lavage and nippleaspirate fluid where the DNA amount is limiting, for example as littleas about 50 to about 100 cells, as well as in larger samples, such asformalin-fixed paraffin-embedded sections of core biopsies. The maximuminput DNA is approximately 600 ng. Using invention methods of QM-MSP,the level and incidence of hypermethylation CpG islands in RASSF1A,TWIST, Cyclin D2, HIN1, and RAR-β, genes in samples where DNA islimiting or when extreme sensitivity is desired can be co-amplified forthe purpose of detecting progression of primary breast cancer in asubject. Scoring the cumulative methylation of these gene promoterswithin a sample gives high sensitivity and specificity of detection ofprimary breast cancer and a global indication of promoterhypermethylation in tumors relative to normal tissue. The inventionmethods are designed to evaluate samples that contain extremely limitedamounts of DNA, such as those from ductal lavage or nipple aspiration.In the process, the extent of gene hypermethylation in primary breastcancer has been evaluated and the method is readily adaptable toclinical testing. Although this technique is illustrated with respect tobreast tissues, the technique can be used to evaluate gene (e.g., genepromoter) hypermethylation within a wide range of tissues.

Recently it has been shown that some of the genes whose promoters aremost frequently hypermethylated (30-90%) in breast carcinomas, but notin normal breast epithelium or circulating blood cells, are Cyclin D2,RARB, TWIST, RASSF1A and HIN1. Widschwendter and Jones (supra) reviewedover 40 genes lost in breast cancer due to promoter hypermethylation. Astudy of 103 cases of breast cancer recently observed that in 100% ofcases of invasive carcinoma, and in 95% of cases of ductal carcinoma insitu (DCIS) one or more gene promoters in a panel of markers consistingof RASSF1A, HIN1, RAR-β, Cyclin D2 and TWIST were hypermethylated. Infact, the vast majority of tumors (80%) have promoters that arehypermethylated for two or more of these five genes, prompting theobservation that tumor tissue may have a pattern of methylation ofmultiple promoters (Fackler M. J. et al. Int J Cancer (2003) in press,online). Thus, it was conceived that profiling the cumulativemethylation of multiple gene promoters associated with a particular typeof cancer, such as primary breast cancer, would serve to betterdistinguish benign from malignant tissues and to provide a more powerfulapproach than characterizing the status of one or more gene markers(Fackler M. J. et al. Cancer Res (2004) 64:4442-4452).

Studies also have demonstrated the feasibility of assessing genepromoter hypermethylation in ductal lavage (DL) samples (Evron E. et al.Lancet (2001) 357:1335-1336). These studies found TWIST, Cyclin D2 orRARB gene promoter hypermethylation in cells derived from subjects withductal carcinoma. However, the status of more than 3 genes in any singlesample could not be assessed because of the limited available DNA. Forexample, the DL samples used in the current study contained 50-1000epithelial cells such that assessment of the status of many genes wouldbe virtually impossible. Therefore, a new strategy was designed tobetter evaluate sources of material where DNA is limited (e.g. ductallavage, plasma, fine-needle or core biopsy, or nipple aspiration fluid).

While QM-MSP was performed in a 96 well platform in the Examplesillustrating the invention, a 384 well platform, or larger can be usedfor high throughput if QM-MSP is formatted for a larger setting. TheQM-MSP technique is applicable to frozen or archival paraffin-embeddedclinical tissues (FIGS. 5-7), as well as ductal lavage material (FIGS.8, 11-12), plasma (data not shown), serum (FIGS. 10-11), nippleaspiration fluid (FIG. 9), and fine needle aspirates (FIGS. 11 and 13).Other sources of sample DNA are also suitable for QM-MSP monitoringincluding but not limited to core tissue biopsies, bronchial washings,buccal cavity washings, cervical scrapings, prostatic fluids, and urine.Conditions that are unrelated to cancer which are suitable formonitoring by the invention include but are not limited to eclampsia andpre-eclampsia (“DNA Methylation Changes in Sera of Women in EarlyPregnancy Are Similar to Those in Advanced Breast Cancer Subjects”Muller H. M. et al. Expert Rev Mol Diagn (2004) 3:443-458.

In a study of 14-28 tissue samples per group, the extent of genepromoter hypermethylation between normal tissues (derived from reductionmammoplasty) and malignant tissues (primary invasive ductal carcinoma)for RASSF1A, TWIST, Cyclin D2, and HIN1 (FIG. 5 and Table 5) was studied(Fackler M. J. et al. Cancer Res (2004) 64:4442-4452). Significantdifferences were observed in the level of promoter hypermethylationbetween normal and tumor tissues for each of these four genes, based oncomparison of mean and median normal values (Table 5).

Importantly, one of the powers of the invention method of QM-MSP is thatit provides a means for defining the normal range of promotermethylation, as shown for RASSF1A, TWIST, HIN1 and Cyclin D2 genes innormal breast tissue (Table 5 and FIG. 5) (Fackler M. J. et al. CancerRes (2004) 64:4442-4452). Techniques that give higher sensitivityusually also give higher “background,” picking up signals that aremissed by other methods. This was also observed in our current study,where a higher incidence of methylation in normal mammoplasty wasobserved than previously (Fackler M. J. et al. Int J Caner (2003) inpress, online) found using the gel-based MSP. Even so, with QM-MSP, themedian of promoter methylation was 0% M for all genes. By setting anupper threshold for normal, the occasional low-level methylation thatoccurs in some normal tissues is acknowledged and criteria are set thatdefine “positive” results as being in tumor. In the current study,cutoffs were established such that approximately 5-10% of normal samplesfell above the cutoff. Using this criterion, the incidence of positiveshowing in tumor tissue was 68% for RASSF1A, 67% for TWIST, 57% forCyclin D2, and 57% for HIN1 (Table 5).

More informative than evaluating the methylation status of a singlegene, studies of cumulative multi-gene promoter hypermethylationindicated that striking differences exist between normal and malignanttissues (FIGS. 6A and 6B)) (Fackler M. J. et al. Cancer Res (2004)64:4442-4452). The panel of four genes (RASSF1A, TWIST, Cyclin D2, andHIN1) was amplified in 9 normal breast tissues and 19 primary carcinomasand the cumulative amount of gene promoter hypermethylation wasdetermined for each of these samples (FIGS. 6A and 6B and Table 6).There was a highly significant (p=0.0002) difference between levels ofcumulative gene promoter hypermethylation in normal tissues and that inmalignant tissues when evaluating RASSF1A, TWIST, Cyclin D2 and HIN1together as a panel. The invention methods employing cumulativemethylation profiling of a panel of four genes was able to detect 84% oftumors; whereas single gene analyses yielded positive results in only57-68% cases, depending on the gene analyzed (FIGS. 5 and 6A-B). To ourknowledge, this result is the first study to describe quantitation ofcumulative methylation of gene promoters associated with a particulartype of carcinoma, and shows its importance in distinguishing betweennormal and tumor tissues (Fackler M. J. et al. Cancer Res (2004)64:4442-4452).

Molecular alterations in histologically normal appearing breast tissueadjacent to tumor have been previously reported, although theirsignificance is not yet clear (Umbricht C. B. et al. Oncogene (2001)20:3348-3353; Deng G. et al. Science (1996) 274:2057-2059; Heid C. A. etal. Genome Res (1996) 6:986-94). In the present study, the cumulativeamount of gene promoter hypermethylation for RASSF1A, TWIST, Cyclin D2,and HIN1 gene promoters was studied in 6 pairings of tumor andapparently histologically normal adjacent tissue (Table 6, FIG. 7). All6 genes showed hypermethylation in tumor tissue. The adjacent normaltissues were also positive for promoter hypermethylation in 4 of 6individuals, although the levels were considerably lower than in thenearby tumor tissue (p=0.01, based on the Mann-Whitney test). Therefore,this study suggests that adjacent normal tissue contains hypermethylatedgenes, albeit at a lower level than the adjacent tumor tissues. Thus,elevated methylation in histologically normal tissue adjacent to tumormay be an indicator the state of tissue cell populations undergoingdisease. The high degree of sensitivity, precision and linearity thatmay be achieved using the invention methods, makes it possible toquantify disease progression, for example, the pre-existence ofmolecular alterations that render the tissue at a higher risk for breastcancer, or are normal age-related changes. It is important to note thatno age-related abnormalities in these genes were observed in studiesconducted as described herein (our unpublished data).

Application of the invention QM-MSP methods to ductal washings showedthat it is possible to co-amplify up to 5 genes (FIG. 8 and Table 7),from limited amounts of DNA (Fackler M. J. et al. Cancer Res (2004)64:4442-4452). Using this approach, it was observed that most genes weresuccessfully amplified in the samples. Analysis for cumulative genepromoter hypermethylation showed that little or no methylation wasdetected in the majority of samples taken from individuals having benignlesions (1 of 8 cases had low level methylation). By comparison, 2 of 4samples with features of ductal carcinoma in situ (DCIS) had extremelyhigh levels of cumulative gene hypermethylation. It can be concludedfrom this analysis that QM-MSP can be applied to analysis of multi-genepromoter hypermethylation in cells derived from washings of breastducts. Furthermore, the QM-MSP method can be applied to investigatemultiple gene promoter hypermethylation that could not have beenevaluated using conventional MSP methods due to limiting quantities ofinput DNA being available.

Of interest, however, 2 of 4 cases of DCIS failed to demonstrate anydetectable methylation when tested according to the invention methods(FIG. 8, Table 7) ) (Fackler M. J. et al. Cancer Res (2004)64:4442-4452). In our previous study of 44 histological sections of DCIStissue, 95% of the samples were hypermethylated for one or more of theRASSF1A, TWIST, Cyclin D2, HIN1, and RARB gene promoters (Fackler M. J.et al. Int J Cancer (2003) in press, online). The reasons for thefailure to detect methylation in all four samples could be that thenumber of cells present in ductal washings was below our sensitivity ofdetection. Conversely, although from the same breast, the washings couldhave been from a duct that was not the one bearing the DCIS. Additionalpromoters associated with breast cancer tissue, APC1, ESR1, BRCA1,BRCA2, P16 and HIC1 already have been added to this multiplex reactionpanel successfully and are undergoing detailed examination (FIGS. 9-13;Primer SEQ ID 46-93) FIGS. 1-2).

The invention is further described by the following example, which ismeant to illustrate, and not to limit, the invention.

Example 1

Materials and Methods

Probes as shown in Tables 1 and 2 above were purchased from AppliedBiosystems (Applied Biosystems, Foster City, Calif.) and other primerswere purchased from Invitrogen (Invitrogen Corporation, Carlsbad,Calif.). Q-MSP primers and probes for β-Actin and methylated Cyclin D2and RASSF1A genes were described in Lehmann et al. (supra). All othersequences were designed in known regions of promoter hypermethylation inbreast carcinoma.

Tissues and cells: Paired primary tumors and adjacent normal tissues(frozen tissue), paraffin-embedded normal breast tissue obtained fromroutine reduction mammoplasty, and primary breast cancer tissues wereobtained from the Surgical Pathology archives of The Johns HopkinsHospital after receiving approval from the institutional review board.The percentage of epithelial cells ranged from 20-50%. Ductal lavage(DL) samples were obtained from the Johns Hopkins CytopathologyLaboratory. Human sperm was obtained from a normal donor. The MDA-MB231breast cancer cell line was obtained from ATCC and cultured as directed.

Sectioning and DNA Extraction from Formalin-Fixed Paraffin-EmbeddedTissue.

The composition of the unstained slides from each archivalformalin-fixed, paraffin-embedded tissue block was confirmed byhistopathological examination of surrounding hematoxylin and eosin (H &E) section. For each tumor, the lesion was identified on an initial H &E section, and confirmed to remain on a serial H & E section takenfollowing preparation of unstained sections for nucleic acid extraction.For DNA extraction, one 5-micron tissue section was deparaffinized inxylene (20 min), scraped from the slide and extracted in 100 μl TNES (10mM Tris, pH 8.0, 150 mM, NaCl, 2 mM EDTA, 0.5% SDS) containing 40 μgproteinase K for 16 hr at 50° C. The tissue extract was heat inactivatedat 70° C. for 10 min and clarified by centrifugation at 14K rpm for 10min; 50 μl of the supernatant was used directly as a source of DNA forsodium bisulfite treatment.

For extraction of DNA from 50-1000 ductal cells, ductal washings werecollected in up to 20 ml of isotonic saline. The cellular suspension wascytocentrifuged onto glass slides and stained with Papanicolaou's stain(Pap Stain) for diagnostic cytological evaluation. The slide coverslipwas then removed by treatment with xylene, and cells were scraped andtransferred to 50 μl TNES containing 40 μg/ml proteinase K, and 200 ngof salmon sperm carrier DNA.

For frozen tissues and MDA-MB231 cells to be used as methylated controlDNA, DNA was extracted with phenol/chloroform using a proceduredescribed in Maniatis T. et al. “Molecular cloning: a laboratory manual”(New York Cold Spring Harbor 1982).

Human sperm DNA (HSD) (unmethylated control) was purified using thePUREGENE® DNA Purification Kit (Gentra Systems, Minneapolis Minn.) withminor modifications to the manufacturer's protocol. Briefly, freshseminal fluid was diluted to 25 ml with TE, and incubated at 37° C. for1 hr. The specimen was centrifuged at 3000 rpm for 15 min at 4° C. Thecellular pellet was rigorously vortexed, resuspended in Cell LysisSolution containing dithiothreitol and proteinase K, and incubated at55° C. overnight. The cell lysate was incubated with RNase A solutionfor 1 hr at 37° C. Proteins were salted out. The DNA was precipitatedwith isopropanol, washed in ethanol, and rehydrated with DNA HydratingSolution (supplied by the manufacturer). HSD was stored at 4° C.

Sodium Bisulfite Treatment of DNA

Tissue, control and cell line DNAs were treated with sodium bisulfiteand analyzed using methylation-specific PCR (MSP) as described by Hermanet al. (supra). This process converts non-methylated cytosine residuesto uracil, while methylated cytosines remain unchanged.Bisulfite-modified samples were aliquoted and stored at −80° C.

Probes and Primers

Quantitative Multiplex Methylation-Specific Polymerase Chain Reaction(MSP)

The QM-MSP procedure required two sequential PCR reactions (FIG. 1). Inthe first PCR reaction (the multiplex step)(RXN 1) 1 μl sodiumbisulfite-treated DNA was added to 24 μl reaction buffer [1.25 mM dNTP,16.6 mM (NH₄)₂SO₄, 67 mM Tris, pH 8.8, 6.7 mM MgC12, 10 mMβ-mercaptoethanol, 0.1% DMSO, and 2.5-5 U Platinum Taq (Invitrogen)]containing 100 ng of each gene specific primer pair (forward and reverseprimer for each of the six gene promoters RASSF1A, RAR-β, TWIST, CyclinD2, HIN1, and β-Actin control)(SEQ ID NOS:1-12). The PCR conditions were95° C. for 5 min, followed by 35 cycles of 95° C. for 30 sec, 56° C. for30 sec, and 72° C. for 45 sec, with a final exten of 72° C. for 5 min.The PCR products were diluted up to 125 μl with water and stored at −20°C. For this QM-MSP reaction, the total input DNA concentration rangedfrom 50 ng purified DNA) to ˜40 pg (for some ductal lavage samples).

For the second reaction (round two of the QM-MSP step)(Rxn 2), 1 μl ofthe diluted PCR product from reaction 1 was used directly, or afterfurther dilution of up to 1:10⁴ (when 50 ng DNA was used in reaction 1).The diluted DNA was added to the real-time—MSP reaction buffercontaining 16.6 mM (NH₄)₂SO₄, 67.0 mM Tris, pH 8.8, 6.7 mM MgCl₂, 10.0mM (β-mercaptoethanol, 0.1% DMSO, 200 μM dNTP, 1.25 U Platinum DNA TaqPolymerase (Invitrogen) and IX ROX (Invitrogen). 600 nM of each of twointernal primers (forward and reverse) (SEQ ID NOS: 13, 14, 16, 17, 19,20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37, 38, 40, 41, 43 and 44)and 200 nM labeled probes (Applied Biosystems) (SEQ ID NOS:15, 18, 21,24, 27, 30, 33, 36, 39, 42 and 45) were also present. The separatereactions were carried out in wells of a 96-well reaction plate in anABI Prism 7900HT Sequence Detector (Applied Biosystems). The reactionsproceeded at 95° C. for 5 min, then 35 cycles of 95° C. for 15 s and60-65° C. (depending on the primer set) for 1 min, with a 10 minextension at 72° C. For each gene included in the reaction plate, thefollowing were used to create standard curves and to providecontrols: 1) serially diluted stock multiplexed HSD/231 DNA (describedbelow, to establish a standard curve), 2) 40,000 copy (40 K) controls,3) no template control and 4) a known DNA (mixed to 1% methylatedcopies; Table 3) to ensure consistency between runs. In addition, 5)100% methylated (MDA-MB231 cell DNA), 6) 0% methylated (HSD DNA, 99-100%unmethylated), and 7) sample lacking template DNA from the first PCRreaction (diluted 1 to 5) were present. All of the above samples wereanalyzed with primer sets for both methylated and unmethylated DNA.

Preparation of Standards.

A stock of multiplexed DNA was prepared as follows: PCR was performed ina reaction mix that contained all six gene primer pairs as well as amixture of 50 ng each of sodium bisulfite treated genomic MDA-MB231 andHSD DNA. Serial dilutions of this stock DNA were used to establish astandard curve in the quantitative real-time PCR reaction (Rxn 2). To dothis, the cycle threshold (C_(T); the cycle in which the signal exceedsthe background) of each dilution was determined during the round twoQM-MSP reaction and then plotted to generate a line for the standardcurve. For each reaction plate, the standards were diluted from the samestock stored frozen at −80° C. for all assays and new dilutions weremade each time.

Copy Number Controls.

For the preparation of this control, unmethylated (U) or methylated (M)genomic DNAs were amplified for each gene in a separate reaction using agene-specific pair of methylation-status specific external primers(Forward, Reverse and Probe) and 50 ng of sodium bisulfite-treatedgenomic DNA derived from either MDA-MB231 (100% methylated) or humansperm DNA (100% unmethylated). A single band was observed by gelelectrophoresis. The reaction products were then purified with aQiaquick® PCR purification kit (Qiagen Inc., Valencia, Calif.), andeluted in 100 μl water. The eluate was quantitated using the NanoDropspectrophotometer (NanoDrop Technologies, Montchanin, Del.) and the DNAconcentration (μg/μl) was determined based on the OD₂₆₀. The molecularweight (μg/μmol) of the PCR product was calculated using BiopolymerCalculator v4.1.1 (C. R. Palmer available on the world wide web at theaddress paris.chem.yale.edu/extinct). Using Avogadro's definition (1μmol=6×10¹⁷ molecules), DNA copies per microliter were calculated: DNAcopies/μl=(DNA concentration)(6×10¹⁷ molecules)/molecular weight. Theconcentration of each gene template control was adjusted to 3×10¹⁰copies per microliter in 1 mg/ml salmon sperm carrier DNA, and stored at−80° C. Unmethylated and methylated DNA for each of the six genes wereobtained and stored separately. A cocktail was then prepared thatcontained 4×10⁶ copies per microliter of each gene in 1 mg/ml salmonsperm DNA. For each reaction plate, the DNA cocktail was then diluted100-fold to 40,000 copies per well. Salmon sperm DNA did not interferewith the PCR reaction (data not shown). A known quantity of DNA (40,000copies per well, denoted “40K” control), prepared as described above,was used to transform the standard curve to represent copy number. Thecycle threshold (C_(T)) of the 40 K control was determined during theQ-MSP reaction and plotted on the line obtained for the standard curve.The copy number for each dilution was then “back calculated,” based onwhere the 40K C_(T) intersected the standard curve. Sample 40 K hadapproximately equal amounts of unmethylated and methylated DNA for eachof the six genes along with carrier salmon sperm DNA (10 μg/ml).

Calculation of % Methylation

The relative amount of methylation for each unknown sample wascalculated as % M=(100)(# copies M/# copies methylated+unmethylatedDNA). To determine the # copies of methylated and unmethylated DNA,sample DNA was mixed with Q-MSP reaction buffer following the multiplexreaction, then assayed with methylated primers and unmethylated primers(in separate wells) in the round two QM-MSP reaction, and a C_(T) wasdetermined for each. Using the ABI Prism SDS 2.0 software supplied byApplied Biosystems with the 7900 HT Sequence Detector, the number ofcopies of methylated and unmethylated DNA was extrapolated from therespective standard curves using the sample C_(T) and applying theabsolute quantification method according to the manufacturer'sdirections. Only values falling within the range covered by the standardcurve were accepted.

Statistical Analysis and Graphical Representation of Data.

Statistical analyses and plotting of data were performed using GraphPadPrism (GraphPad Software Inc., San Diego, Calif.). P values<0.05 wereconsidered significant and all tests were two-tailed. The nonparametricMann-Whitney test was used to test whether the samples are fromidentical distributions, indicating their medians are equal. Samplemeans were compared using the unpaired t test, assuming unequalvariances (Welch's correction). For testing of means, data weretransformed as a function of Ln_(e) (% M+1) where stated to fulfill theassumption of normality. The Fisher's exact test was used to testwhether the differences between the incidence of positivity formethylation in tumor and normal were significant.

Results

Quantitative-Multiplex PCR (QM-MSP) Assay is a Two-Step Reaction:

Validation of the QM-MSP Assay-Multiplex PCR, the first step in theQM-MSP assay, was accomplished by performing two sequential PCRreactions as shown schematically in FIG. 1). In the first PCR reaction,a cocktail of six pairs of gene-specific primers were used to co-amplifyCyclin D2, RARB, TWIST, RASSF1A, HIN1, and Actin (as a control) (Tables1 and 2). These external primer pairs were complimentary to thesequences flanking the CpG islands that were to be queried in the secondPCR reaction. External primers were selected to exclude CGdinucleotides, thereby rendering DNA amplification independent of themethylation status.

Both U and M Primers are Equally Efficient in Amplifying DNA—Primer setsspecifically for methylated (M) and unmethylated (U) DNA were designedfor comparable performance; to confirm this, the delta C_(T) (C_(T)M-C_(T) U) was plotted as a function of sample dilution over a widerange of dilutions (10⁻³ to 10⁻⁸) of the standard stock HSD/231 DNA.Analyses were performed as is shown for RASSF1A in FIGS. 2A-B. The deltaC_(T) was approximately the same for all dilutions, as shown by thehorizontal nature of the line, indicating that the primer sets wereequally efficient over 5 logs of template quantities (FIG. 2A). Inaddition, for both unmethylated and methylated standard curves theslopes are approximately −3.33, which is reflective of a two-foldincrease in PCR product per cycle during the linear phase of real-timePCR. Finally, a correlation coefficient (R2) of 0.999 indicates thatlinearity exists over the entire range of template concentration asshown in FIG. 2B). Similar results were obtained for the other fivegenes in our study (data not shown).

Specificity and Sensitivity—To assess the sensitivity and specificity ofthe QM-MSP method, a mixing experiment was performed usingcolumn-purified, PCR-amplified fragments of sodium bisulfite-modifiedDNA as template as shown in Table 3 below. The amount of unmethylatedDNA was kept constant (100,000 copies per well) and the amount ofmethylated DNA was decreased (1000-0.1 copy per well). Although thegeneral efficiency of real time PCR is known to fall off at less than100 copies, it was expected that some level of methylation would bedetected below 100 copies, although probably not in a linear manner. ForRASSF1A and TWIST, methylation was detected at 1 methylated in 100,000unmethylated copies and for Cyclin D2 and HIN1 at 10 methylated in100,000 unmethylated copies. Therefore, the overall sensitivity of themethod is 1-10 in 100,000.

TABLE 3 Determination of the sensitivity of QM-MSP. % Methylation^(c)RASSF1A Twist Hin-1 Cyclin D2 # Copies M and U Template^(a) 1000/100,000 0.9220 2.7604 4.8024 1.5760   100/100,000 0.0602 0.24820.6273 0.1525   10/100,000 0.0078 0.0272 0.0024 0.0025    1/100,0000.0072 0.0012 0.0000 0.0000   .1/100,000 0.0000 0.0000 0.0000 0.0000 HSD(100% U control) 0.0000 0.0003 0.0000 0.0000 231 (100% M control)99.9981 100.0000 100.0000 100.0000 Water 0.0000 0.0000 0.0000 0.0000#Copies M and U^(b)   300/29700 0.7823 1.2190   30/2970 0.7195 0.9376   3/297 0.2390 0.1451 ^(a,b)Purified methylated (M) and unmethylated(U) stock DNA template were mixed in the proportions indicated.^(a)Unmethylated DNA was kept constant as the amount of methylated DNAwas decreased. ^(b)A 1% M control was kept constant as the totalquantity of DNA decreased. ^(c)The % Methylation of RASSF1A, TWIST, HIN1and Cyclin D2 from the QM-MSP assay is shown. The assay sensitivity is1-10 in 100,000 for methylated DNA and detects as few as 1-3 copies ofmethylated DNA.

The highly specific performance of the methylated primers wasdemonstrated using the HSD control (100% unmethylated DNA), and0.1/100,000 sample (diluted to <1 copy of methylated in the presence of100,000 copies of unmethylated DNA), both of which showed 0% M in theQM-MSP reaction (Table 3, FIG. 3). Likewise, no unmethylated signal (0%U) was detected in MDA-MB231 methylated DNA control (100% methylatedDNA). It was expected that the 1000/100,000 sample would be read as 1%methylated (1000 copies of methylated and 100,000 copies ofunmethylated). That some samples were slightly higher (e.g. 4.8% forHIN1, 2.8% for TWIST) probably reflects difficulty in accuratelydiluting samples from a starting concentration of 3×10¹⁰ copies per μl(diluted over 5-10 logs).

To characterize the behavior of the QM-MSP assay below the lower limitof linearity (specifically, whether a 1% M template produces a 1% Massay result as the total quantity of DNA template diminishes), a “1%”standard was serially diluted and samples were tested beginning at atemplate quantity estimated to contain 300 copies of methylated DNA to29700 unmethylated copies (300/29700), using column-purified DNA as atemplate (Table 3). The lowest quantity tested contained three copiesmethylated to ˜297 copies unmethylated DNA (3/297). The results forRASSF1A and TWIST (Table 3) revealed that at ratios of 300/29700 and30/2970 total copies, the assay result (% M) was approximately 1% foreach. Methylation was still detectable at the lowest ratio templatequantity tested, at three copies of methylated DNA, consistent with theprevious experiment (1 copy of methylated DNA was detected in 100,000copies of unmethylated DNA). We found a bias towards underreporting ofthe % M below 30 copies of methylated DNA, probably reflecting therelative lack of efficiency of the methylated reaction compared to theunmethylated reaction that contained nearly 100-fold more copies of thegene (Table 3). This result was predicted, since linearity is generallyknown to be lost below 100 copies of DNA template in real-time PCR.

Genomic DNA is a more challenging template than PCR-amplified DNAbecause breakage of genomic DNA is known to occur in the process ofsodium bisulfite conversion. To evaluate the sensitivity of the QM-MSPmethod with genomic DNA, DNA mixing experiments were performed withapproximately 40 pg methylated DNA (˜13 copies derived from 231 cellDNA) and 600-60000 pg unmethylated genomic DNA (˜200-20,000 copies ofHSD). Using the QM-MSP method, 40 pg of methylated RASSF1A genomic DNAwas easily detected even in the presence of a 1500-fold excess ofunmethylated DNA as shown in FIG. 4), when the conversion estimate of 6pg/2 copies/cell of DNA was used.

Comparison Between QM-MSP and Q-MSP

It is possible that performing a two-step multiplex PCR method couldyield results that differed from those obtained with a direct one-stepPCR method because of the addition of the multiplex step. We performedQM-MSP and direct Q-MSP assay on a panel of five tumor DNAs andcalculated the percentage of methylation by the (U+M) method to estimatetotal DNA. With few exceptions, there was excellent concordance betweenthe percentage of methylation values obtained for the RASSF1A, TWIST,HIN1 or Cyclin D2 genes (Table 4).

TABLE 4 Comparison between Q-MSP and QM-MSP % M Q- QM- MSP MSP RASSF1A 195 99 2 80 81 3 39 41 4 12 21 5 28 34 HSD 0 0 231 100 100 Water 0 0TWIST 1 96 70 2 0 0 3 54 26 4 1 3 5 0 5 HSD 0 0 231 100 100 Water 0 0HIN1 1 95 82 2 81 74 3 82 48 4 41 16 5 47 43 HSD 0 0 231 100 100 Water 00 Cyclin D2 1 47 25 2 0 0 3 1 0 4 19 22 5 2 0 HSD 0 0 231 100 100 Water0 0

The percentage of methylation (% M) was calculated as: (the number ofcopies of methylated DNA divided by the number of copies of unmethylatedDNA+methylated DNA)×100, using absolute quantitation. The positivecontrol for unmethylated DNA was human sperm DNA (HSD), and formethylated DNA was MDA MB231 (231).

Quantitation of Methylation in Breast Carcinoma and Comparison to NormalBreast Tissue

Paraffin-embedded, formalin-fixed tissues from routine reductionmammoplasty (14 to 28 samples each) and frozen primary breast carcinomatissues (19 to 21 samples each) were analyzed by QM-MSP for genepromoter hypermethylation of RASSF1A, TWIST, Cyclin D2, and HIN1 (Table5, FIGS. 5A-B). Head to head comparison of DNA extracted from frozen orfixed tissue showed no significant differences in methylation (data notshown). RASSF1A hypermethylation ranged from 0-71% (mean=18.5%) intumors and 0-56% (mean=2.6%) in normal tissues (p=0.0001); TWISThypermethylation ranged from 0-72% (mean=21.1%) in tumors and 0-1.6%(mean=0.11%) in normal tissues (p=0.0001); HIN1 hypermethylation rangedfrom 0-82.2% (mean=24.5%) in tumors, and 0-18% (mean=2.3%) in normaltissues (p=0.003); and Cyclin D2 hypermethylation ranged from 0-44.5%(mean=5.0%) in tumors, and 0-0.2% (mean=0.02%) in normal tissues(p=0.02). Highly significant differences in the medians were alsopresent for all genes, RASSF1A (p=0.0001), TWIST (p=0.001), HIN1(p=0.003), and Cyclin D2 (p=0.0009) (using the Mann-Whitney test onuntransformed data).

A laboratory cutoff (% M) was established for each gene such that about90-95% of normal breast tissues would be at or below the cutoff (Table5). Using the cutoff of 2% M for RASSF1A and HIN1, 0.5% for TWIST, and0.2% for Cyclin D2 in normal tissues, values above the cutoff wereconsidered “positive” for hypermethylation. Among tumors, 68% (13 of 19)were positive for RASSF1A, 67% (14 of 21) for TWIST, 57% (12 of 21) forCyclin D2, and 57% (12 of 21) for HIN1. By comparison, 7% (2 of 28) ofnormal mammoplasty samples were positive for RASSF1A, 6% (1 of 18) forTWIST, 7% (1 of 16) for Cyclin D2, and 14% (2 of 14) for HIN1. Somesamples had low-level methylation that was below the cutoff. Using 0% Mas a cutoff for each gene, tumor vs. normal hypermethylation was 89%positive vs. 7% for RASSF1A, 68% vs 17% for TWIST, 71% vs. 12% forCyclin D2, and 76% vs. 21% for HIN1. Using these cutoffs, a significantdifference in the incidences of positivity between tumor and normaltissues was observed (RASSF1A p<0.00002, TWIST p<0.0002, CyclinD2p<0.002, and HIN1 p<0.02; Fisher's exact).

TABLE 5 Normal versus Malignant Breast Tissues - Quantitation of theLevel of Gene Promoter Hypermethylation Positive for Upper 95%methylation^(a) Range % Median % Mean ± SEM % Lower 95% CI % CI % # (%)Normal Breast RASSF1A 0-56 0^(b) 2.6 ± 2.0 (0)    6.7  2/28 (7) TWIST0-1.6 0^(c) 0.11 ± 0.09 (0)    0.29  1/18 (6) HIN1 0-18 0^(d) 2.3 ± 1.5(0)    5.5  2/14 (14) Cyclin D2 0-0.2 0^(e) 0.019 ± 0.014 (0)    0.48 1/16 (7) Breast Carcinoma RASSF1A 0-71 7.0^(b) 18.5 ± 4.7  8.7 28.213/19 (68) TWIST 0-72 5.0^(c) 21.1 ± 5.5  9.6 32.6 14/21 (67) HIN1 0-829.9^(d) 24.5 ± 6.1  11.8  37.3 12/21 (57) Cyclin D2 0-44 0.26^(e) 5.0 ±2.5 (0)    10.3 12/21 (57) ^(a)Based on cutoffs of 2% M for RASSF1A andHIN1, 0.5% M for TWIST, and 0.2% M for Cyclin D2; ^(b)p = 0.0001 forRASSF1A, ^(c)p = 0.001 for TWIST, ^(d)p = 0.003 for HIN1, and ^(e)p =0.0009 for Cyclin D2.Cumulative Gene Promoter Hypermethylation Scores in Primary BreastCancer

To determine the cumulative amount of gene promoter hypermethylation,QM-MSP was performed and the sum of all % M within the panel of geneswas used to provide an overall cumulative score for each sample. Thiswas represented graphically relative to MDA-MB231 DNA, which is 100%methylated for RASSF1A, TWIST, Cyclin D2 and HIN1. Therefore, thiscontrol DNA has a relative score of 400 in FIG. 6A. The cumulativemethylation profiles of nine normal mammoplasty samples were compared tothose of 19 invasive tumors (Table 6; FIG. 6). Normal tissues rangedfrom 0-18 units, and tumors ranged from 1-248. Among the nine normaltissues tested for four genes (36 values) the cumulative score had amean of 2.61±2.05 (median=0) (FIG. 6B). Among the 19 tumors tested forfour genes (76 values) the cumulative score had a mean of 72.8±15.03units (median=74) out of a possible 400 units (as above, Table 6 andFIGS. 6A and 6B). The difference in log-transformed means between normaland malignant breast tissue was highly significant (p-0.0001, unpaired ttest with Welch's correction).

We explored the use of 4.7 units as a cumulative score cutoff value.This was based on the individual gene cutoffs above (2% each for RASSF1Aand HIN1, 0.5% for TWIST and 0.2% for Cyclin D2=4.7%). Using thiscutoff, 84% (16) tumor samples were positive. Although 3 of 19 tumorsfell below the cutoff, all of the “negative” tumors were methylated atlow levels for one or more genes in this panel (FIG. 6A). Among normalsamples, 89% (8) were negative. Thus in this group of 28 samples (9normal and 19 tumor), the sensitivity of detection of tumor was 0.84 andspecificity was 0.89, an overall accuracy of 0.86 (24 of 28)

TABLE 6 Cumulative promoter hypermethylation in normal, adjacent“normal” and malignant breast tissues. Lower Upper Positive Range MedianMean + SEM 95% CI 95% CI for methylation^(b) Tissue Units^(a) Units^(a)Units^(a) units^(a) units^(a) # (%) n Normal 0-18  0^(c)  2.61 ±2.05^(d) (0)  7.35  1/9 (11) 9 Carcinoma 0-248  74^(c)  72.8 ± 15.03^(d) 41.3 104.4 16/19 (84) 19 Normal Mammoplasty and Malignant BreastTissues Comparison of Paired Tumor and Adjacent Normal Breast EpitheliumLower Upper Positive Range Median Mean + SEM 95% CI 95% CI formethylation^(§) Tissue Units* Units* Units* units* units* # (%) nAdjacent 2-29  9^(e)  11.7 ± 4.07  1.2 22.1  4/6 (67) 6 “Normal”Carcinoma 5-258 133^(e) 129.2 ± 39.9 26.5 231.8  6/6 (100) 6^(a)Relative units of methylation is the sum of percent methylation foreach of four genes in the panel; ^(b)Based on a cutoff of ≦4.7 units.^(c)p = 0.0001, ^(d)p = 0.0002; ^(e)p = 0.03.

In an independent experiment, six pairs of tumor and adjacent tissuefrom the surgical margins, which were histologically normal, wereexamined to determine the cumulative amount of gene promoterhypermethylation in RASSF1A, TWIST, Cyclin D2, and HIN1 (Table 65, FIG.7). The cumulative methylation ranged from 2-29 units within adjacentnormal tissues and 5-258 units within tumor tissues, out of a possible400 units (FIG. 7). Using the cutoff established for cumulative normalin mammoplasty samples (<4.7 units, see above), all six tumors werepositive. The adjacent “normal” tissues were also positive in four ofsix individuals. While the cumulative methylation levels within adjacent“normal” tissues were significantly lower than the nearby tumor (p=0.03by Mann-Whitney), the tumor-adjacent tissues had a significantly higherlevel of methylation than normal mammoplasty samples (p=0.01 byMann-Whitney; Table 6 and FIG. 7).

Detection of Multi-Gene Promoter Hypermethylation in Breast Duct Cells

To test the applicability of this test to small clinical samples, weextracted DNA from ductal lavage (DL) cells that had been scraped fromcytospin preparations after cytological evaluation and performed QM-MSPto test for RASSF1A, TWIST, Cyclin D2, RAR-β, and HIN1 gene promoterhypermethylation (Table 7, FIG. 8). The total number of epithelial cellspresent in the samples varied from 50-1000. The level of gene promotermethylation was quantitated for each sample and the cumulative promotermethylation profile was established. High level hypermethylation wasdetected in two of four cases of DCIS. Among diagnostically benignsamples, six of seven showed no cumulative hypermethylation at all,compared to one of seven which revealed low level RASSF1A methylation(Table 7 and FIG. 8).

TABLE 7 Quantitative multiplexed methylation-specific PCR analysis ofductal breast cells % Methylation ID RASSF1A TWIST Cyclin D2 HIN1 RARBDuctal Lavage Cells From High Risk Women^(a) Cytology Mammography 1 0 00 0 0 Benign Normal 2 0 0 0 0 0 Benign Normal 3 0 0 0 0 0 Benign Normal4 0 0 0 0 0 Benign Normal 5 0 0 0 0 0 Benign Normal 6 0 0 0 0 0 BenignNormal 7 0.4 0 0 0 0 Benign Normal Ductal Lavage Cells From Women WithCarcinoma^(b) Cytology Histopathology 8 0 0 0 0 0 Benign DCIS 9 0 0 0 00 Benign DCIS 10 4 0 12 75 0 Inadequate Invasive Carcinoma 11 47 31 4 6589 Markedly atypical Invasive Carcinoma

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Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. A method for determining an increased likelihood of development ofbreast cancer associated with aberrant methylation of DNA in a subject,comprising: a) co-amplifying DNA in a single subject sample comprising aplurality of different DNA sequences isolated from the subject sampleusing a mixture of DNA sequence-specific, methylation status-independentouter primer pairs that selectively hybridize to three or more of theDNA sequences under conditions that allow generation of a firstamplification product containing first amplicons, wherein the subjectsample is selected from the group consisting of ductal lavage fluid,nipple aspiration fluid, breast tissue, breast tumor biopsy, and fineneedle aspirates, and wherein further the DNA sequences comprise threeor more genes selected from the group consisting of RASSF1A, TWIST,HIN1, and Cyclin D2; b) co-amplifying the first amplicons by real-timePCR under conditions that allow generation of three or more secondamplification products; adding three or more members of a set of DNAsequence-specific probes comprising an optically detectable label andthree or more members of a set of DNA sequence-specific methylationstatus-dependent inner primer pairs, wherein the probes and the innerprimer pairs selectively hybridize cognate first amplicons, and whereinthe sets of probes and inner primer pairs collectively bind to aplurality of different first amplicons in the first amplificationproduct; c) detecting signal intensity of the optical label in thesecond amplification products to determine the amount of methylation ofthe second amplification products; and d) deriving a combinedmethylation value for the methylation in the subject sample from amountsof methylation determined for the second amplification products ascompared with a combined methylation value in comparable normal tissueto diagnose the state of development of breast cancer in the subject. 2.The method of claim 1, wherein d) comprises determining a total detectedsignal intensity from the second amplification gene products to measurea first amount of gene promoter hypermethylation in the tissue of thesubject as compared with a second amount of methylation in comparablenormal tissue to diagnose the state of development of the condition inthe subject.
 3. The method of claim 1, wherein the probes are molecularbeacons or bilabeled oligonucleotide probes comprising a fluorescentmoiety and a quencher moiety.
 4. The method of claim 1, wherein thesubject is a human.
 5. The method of claim 1, wherein the signalintensities from the second amplification products are detected seriallyor simultaneously.
 6. A method for determining an increased likelihoodof development of breast cancer associated with hypermethylation of CpGislands in cancer-associated tissue of a subject, comprising: a)co-amplifying CpG islands in a single subject sample of acancer-associated tissue comprising a plurality of different DNAsequences isolated from the carcinoma-associated tissue using a mixtureof three or more DNA sequence-specific, methylation status-independentouter primer pairs that selectively hybridize to cognate DNA sequencesunder conditions that allow generation of a first amplification productcontaining first amplicons, wherein the cancer-associated tissue isselected from the group consisting of ductal lavage fluid, nippleaspiration fluid, breast tissue, breast tumor biopsy, and fine needleaspirates, and wherein further the DNA sequences comprise three or moregenes selected from the group consisting of RASSF1A, TWIST, HIN1, andCyclin D2; b) using real time PCR to co-amplify CpG islands in the firstamplicons under conditions that allow generation of three or more secondamplification products, using three or more members of a set of DNAsequence-specific probes comprising three or more fluorescent moietiesand three or more members of a set of DNA sequence-specific methylationstatus-dependent inner primer pairs that selectively hybridize to threeor more first amplicons, wherein the sets of probes and inner primerpairs collectively hybridize to a plurality of different first ampliconsin the first amplification product; c) detecting fluorescence due to thepresence of the fluorescent moieties in the second amplificationproducts to determine the amount of methylation of the CpG islandstherein; and d) deriving a combined methylation value for the CpGislands in the DNA sequences from amounts of methylation determined forthe second amplification products as compared with the combinedmethylation value in a comparable normal tissue sample to diagnose thestate of development of breast cancer in the subject.
 7. The method ofclaim 6, wherein d) comprises determining cumulative detectedfluorescence from the second amplification products to measure acumulative amount of gene promoter hypermethylation in the DNA of thetissue of the subject as compared with a cumulative amount ofmethylation in the DNA of comparable normal tissue to diagnose the stateof development of the cancer in the subject.
 8. The method of claim 6,wherein fluorescence is serially or simultaneously detected in c) fromthe second amplification products.
 9. The method of claim 6,additionally comprising adding an internal standard in b) for assessingrelative amounts of hypermethylation in CpG islands in the DNA sequencesafter amplification.
 10. The method of claim 6, wherein the combinedmethylation value of the CpG islands is correlated to the development ofthe cancer in the subject by comparing the amount of amplification ofthe CpG islands to the amount of amplification product formed from knownstandards.
 11. The method of claim 10, wherein the standards areMDA-MB231 cell DNA and human sperm DNA or similarly methylated orunmethylated DNA.
 12. The method of claim 6, wherein the amplificationin b) is performed by cyclic polymerase-mediated reaction using Taq DNApolymerase.
 13. The method of claim 6, wherein the DNA sequencescomprise a regulatory region.
 14. The method of claim 13, wherein theregulatory region is a promoter.
 15. The method of claim 6, wherein thesubject sample comprises as few as about 50 to about 1000 carcinomacells.
 16. The method of claim 6, wherein the first amplificationproduct is diluted prior to b).
 17. The method of claim 16, whereindilution is in the range from 1:5 to 1:10⁴.
 18. The method of claim 6,further comprising using the level of unmethylated CpG island in thesecond amplification products as the internal control for assessing theextent of methylated CpG island present.
 19. The method of claim 6,wherein the subject sample comprises as few as about 50 to about 1000tumor cells.
 20. The method of claim 6, wherein fluorescence isseparately detected from the fluorescent moieties in separate aliquotsof the first amplification product to indicate methylation status of thesecond amplicons in the separate aliquots.
 21. The method of claim 6,wherein the comparable normal tissue is obtained from the subject. 22.The method of claim 6, wherein the comparable normal tissue is obtainedfrom apparently normal tissue adjacent to the source of thetumor-related tissue.
 23. The method of claim 6, wherein the subject isa human.
 24. The method of claim 6, wherein the signal intensities fromthe second amplification products are detected serially orsimultaneously.
 25. The method of claim 6, wherein the DNA sequence is agene.
 26. The method of claim 6, wherein the first amplification productis diluted prior to b).
 27. The method of claim 26, wherein the dilutionis in the range from 1:5 to 1:10⁴.
 28. The method of claim 6, whereinthe subject sample is ductal lavage fluid.
 29. The method of claim 6,wherein the isolated DNA sequences are selected from RASSF1A, TWIST,Cyclin D2, and HIN1 promoters.
 30. The method of claim 29, wherein thefirst outer primer pairs in a) are selected from SEQ ID NOS: 1-6 and9-10.
 31. The method of claim 6, wherein in b) members of the set ofsecond primer pairs selectively hybridize to first amplicons containingmethylation of a CpG island and b) is repeated using an additional setof second primer pairs that selectively hybridize to ampliconscontaining unmethyation of a CpG island in the first amplicons.
 32. Themethod of claim 30, wherein the second inner primer pairs in b) areselected from DNA sequences having nucleotide sequences of SEQ ID NOS:13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 37, 38, 40 and 41, andthe probes are selected from DNA sequences having nucleotide sequencesof SEQ ID NOS: 15, 18, 21, 24, 27, 30, 39 and
 42. 33. The method ofclaim 6, further comprising using levels of unmethylated secondamplicons for each DNA sequence as an internal control for assessing theextent of methylated second amplicon present.
 34. The method of claim 6,wherein as few as 1 to about 10 methylated copies of DNA in a mixturecontaining up to about 100,000 copies of unmethylated DNA is detected.35. The method of claim 6, wherein as little as about 40 pg ofmethylated genomic DNA in up to about 1500-fold excess of unmethylatedDNA is detected.
 36. The method of claim 6, wherein a predisposition tothe condition in the subject is diagnosed.
 37. The method of claim 6,wherein the plurality of DNA sequences is about three to about sevengenes per set which are co-amplified in round one PCR and where it ispossible to amplify two or more sets of genes from picograms of inputDNA.
 38. A method for determining the methylation status of a DNA samplecomprising: a) co-amplifying a plurality of DNA sequences obtained froma subject sample using a mixture of DNA sequence-specific, methylationstatus-independent outer primer pairs that selectively hybridize tothree or more of the DNA sequences under conditions that allowgeneration of a first amplification product containing first amplicons,wherein the DNA sequences comprise three or more genes selected from thegroup consisting of RASSF1A, TWIST, HIN1, and Cyclin D2; b) usingquantitative PCR to amplify first amplicons under conditions that allowgeneration of second amplification products, using a set of DNAsequence-specific, methylation status-dependent inner primer pairs and aset of DNA sequence-specific probes comprising one or moredistinguishable optically detectable labels, wherein a combination ofinner primer pair and probe selectively hybridizes to one firstamplicon, and wherein the sets of inner primer pairs and probescollectively hybridize to a plurality of first amplicons in the firstamplification product; c) detecting signal intensities of the one ormore distinguishable labels in the second amplification products todetermine the amount of methylation of the second amplificationproducts; and d) deriving a combined methylation value for themethylation in the DNA sequences from amounts of methylation determinedfor the second amplification products as compared with the combinedmethylation value in a comparable normal DNA sample to determine themethylation status of the DNA sample.
 39. A method for determining anincreased likelihood of development of breast cancer associated withaberrant methylation of DNA in a subject, comprising: a) co-amplifyingDNA in a single subject sample comprising a plurality of different DNAsequences isolated from the subject sample using a mixture of DNAsequence-specific, methylation status-independent outer primer pairsthat selectively hybridize to three or more of the DNA sequences underconditions that allow generation of a first amplification productcontaining first amplicons, wherein the subject sample is selected fromthe group consisting of ductal lavage fluid, breast tissue and breasttumor biopsy, and wherein further the DNA sequences comprise three ormore genes selected from the group consisting of RASSF1A, TWIST, HIN1,and Cyclin D2, and wherein further the first external primer pairs in a)are selected from SEQ ID NOS:1-6 and 9-10; and b) co-amplifying thefirst amplicons by real-time PCR under conditions that allow generationof three or more second amplification products using three or moremembers of a set of DNA sequence-specific probes comprising an opticallydetectable label and three or more members of a set of DNAsequence-specific methylation status-dependent inner primer pairs,wherein the probes and the inner primer pairs selectively hybridizecognate first amplicons, and wherein the sets of probes and inner primerpairs collectively bind to a plurality of different first amplicons inthe first amplification product; c) detecting signal intensity of theoptical label in the second amplification products to determine theamount of methylation of the second amplification products; and d)deriving a combined methylation value for the methylation in the subjectsample from amounts of methylation determined for the secondamplification products as compared with a combined methylation value incomparable normal tissue to diagnose the state of development of breastcancer in the subject.
 40. The method of claim 39, wherein d) comprisesdetermining a total detected signal intensity from the secondamplification gene products to measure a first amount of gene promoterhypermethylation in the tissue of the subject as compared with a secondamount of methylation in comparable normal tissue to diagnose the stateof development of the condition in the subject.
 41. The method of claim39, wherein the three or more members of the set of DNAsequence-specific methylation status-dependent inner primer pairs in b)have DNA sequences selected from SEQ ID NOS: 13, 14, 16, 17, 19, 20, 22,23, 25, 26, 28, 29, 37, 38, 40 and
 41. 42. The method of claim 39,wherein the three or more members of the set of DNA sequence-specificprobes in b) have DNA sequences selected from SEQ ID NOS: 15, 18, 21,24, 27, 30, 39 and
 42. 43. The method of claim 39, wherein the subjectis a human.